U.S. patent application number 16/032442 was filed with the patent office on 2019-01-17 for kinase inhibitors for treatment of disease.
The applicant listed for this patent is BioAxone BioSciences, Inc.. Invention is credited to Matthew D. ABBINANTI, Lisa MCKERRACHER, Kenneth M. ROSEN, Joerg RUSCHEL.
Application Number | 20190015404 16/032442 |
Document ID | / |
Family ID | 65000451 |
Filed Date | 2019-01-17 |
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United States Patent
Application |
20190015404 |
Kind Code |
A1 |
RUSCHEL; Joerg ; et
al. |
January 17, 2019 |
KINASE INHIBITORS FOR TREATMENT OF DISEASE
Abstract
Disclosed are therapeutic compositions including BA-1076 and/or
BA-2057, methods of their use in the treatment of ophthalmological
disorders. The therapeutic compositions may further include an
IOP-lowering prostaglandin. The methods may further include
administration of an IOP-lowering prostaglandin.
Inventors: |
RUSCHEL; Joerg; (Cambridge,
MA) ; ABBINANTI; Matthew D.; (Westford, MA) ;
ROSEN; Kenneth M.; (Milton, MA) ; MCKERRACHER;
Lisa; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioAxone BioSciences, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
65000451 |
Appl. No.: |
16/032442 |
Filed: |
July 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62531322 |
Jul 11, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61P 27/06 20180101; A61K 9/0019 20130101; A61K 31/4725 20130101;
A61K 31/5575 20130101; A61K 45/06 20130101; A61K 9/0048 20130101;
A61K 9/0053 20130101; A61K 31/5575 20130101; A61K 2300/00 20130101;
A61K 31/4725 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/4725 20060101
A61K031/4725; A61K 9/00 20060101 A61K009/00; A61P 27/06 20060101
A61P027/06 |
Claims
1. A therapeutic composition comprising a therapeutically effective
amount of a compound of formula: ##STR00009## or a pharmaceutically
acceptable salt thereof, wherein BA-1076 or a pharmaceutically
acceptable salt thereof is present in at least 10% enantiomeric
excess.
2. The therapeutic composition of claim 1, further comprising a
therapeutically effective amount of a compound of formula:
##STR00010## or a pharmaceutically acceptable salt thereof, wherein
BA-2057 or a pharmaceutically acceptable salt thereof is present in
at least 10% enantiomeric excess.
3. A therapeutic composition comprising a therapeutically effective
amount of a compound of formula: ##STR00011## or a pharmaceutically
acceptable salt thereof, wherein BA-2057 or a pharmaceutically
acceptable salt thereof is present in at least 10% enantiomeric
excess.
4. The therapeutic composition of claim 3, wherein BA-2057 is
present in at least 50% enantiomeric excess.
5-6. (canceled)
7. The therapeutic composition of claim 1, wherein BA-1076 is
present in at least 50% enantiomeric excess.
8-9. (canceled)
10. The therapeutic composition of claim 1, further comprising
latanoprost, travaprost, bimatoprost, or tafluprost.
11. (canceled)
12. The therapeutic composition of claim 1, wherein the therapeutic
composition is formulated for ocular topical administration,
intravitreal administration, intraocular administration, retinal
administration, oral administration, or intravenous
administration.
13. The therapeutic composition of claim 12, wherein the
therapeutic composition is in a dosage form of eye drops,
formulated for oral administration, or formulated for intravenous
administration.
14. The therapeutic composition of claim 1, wherein the therapeutic
composition comprises the compound at a concentration of 0.001% to
5% (w/v) or at a dose of 0.01 mg/kg to 10 mg/kg.
15-18. (canceled)
19. A method of treating glaucoma, retinitis pigmentosa, macular
degeneration, retinal angiogenesis, corneal blindness, Fuchs'
corneal dystrophy, or corneal scarring in a subject in need
thereof, comprising administering to the subject a
therapeutically-effective amount of the therapeutic composition of
claim 1.
20. The method of claim 19, wherein the method is for treating
glaucoma, retinitis pigmentosa, macular degeneration, retinal
angiogenesis, corneal blindness, Fuchs' corneal dystrophy, or
corneal scarring in the subject.
21-27. (canceled)
28. The method of claim 19, wherein the therapeutic composition is
administered topically, intravitreally, intraocularly, retinally to
the eye, orally, or intravenously.
29-31. (canceled)
32. The method of claim 19, wherein the method further comprises
administering travaprost, bimatoprost, latanoprost, or
tafluprost.
33. (canceled)
34. The method of claim 19, wherein the method of treating corneal
scarring comprises reducing post-operative corneal scarring in a
subject in need thereof.
35. The therapeutic composition of claim 3, further comprising
latanoprost, travaprost, bimatoprost, or tafluprost.
36. A method of treating glaucoma, retinitis pigmentosa, macular
degeneration, retinal angiogenesis, corneal blindness, Fuchs'
corneal dystrophy, or corneal scarring in a subject in need
thereof, comprising administering to the subject a
therapeutically-effective amount of the therapeutic composition of
claim 3.
37. The method of claim 36, wherein the method is for treating
glaucoma, retinitis pigmentosa, macular degeneration, retinal
angiogenesis, corneal blindness, Fuchs' corneal dystrophy, or
corneal scarring in the subject.
38. The method of claim 36, wherein the therapeutic composition is
administered topically, intravitreally, intraocularly, retinally to
the eye, orally, or intravenously.
39. The method of claim 36, wherein the method further comprises
administering travaprost, bimatoprost, latanoprost, or
tafluprost.
40. The method of claim 36, wherein the method of treating corneal
scarring comprises reducing post-operative corneal scarring in a
subject in need thereof.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel kinase inhibitors for
treatment of diseases of the nervous system, ophthalmological
indications and gastrointestinal disorders.
BACKGROUND
[0002] Rho kinase (ROCK) is a kinase found in all eukaryotic cells.
It regulates key processes that include cell motility, cell
differentiation, cell survival, cell-cell junctions and expression
of extracellular matrix proteins. There are two isoforms of ROCK,
ROCK1 and ROCK2. ROCK2 is more highly expressed in the CNS. It is
also the form most highly expressed in tissues that have
dysregulated ROCK in disease. Therefore ROCK-2 selective inhibitors
may be used to treat a variety of diseases accompanied by abnormal
or pathological activation of ROCK signaling for instance
inflammatory stimuli, the microbiome or other factors that increase
the activity of ROCK2 leading to progression of disease.
[0003] The Abl tyrosine kinase was identified as a critical driver
of leukemia from studies of the Abelson murine lymphosarcoma virus
that induced cellular transformation and lymphomas. Subsequent
studies demonstrated that chromosomal translocation of ABL1 to the
breakpoint cluster region (BCR) gene sequences results in
production of the BCR-ABL1 fusion protein and elevated tyrosine
kinase activity in patients with Philadelphia (Ph)
chromosome-positive human leukemia. Subsequently studies of Abl
show that, like ROCK, it regulates many cellular processes leading
to disease. Abl is not typically active in neurons, but is
activated in many neurological diseases. Abl regulates diverse
cellular processes and can be activated by multiple stimuli leading
to cytoskeletal reorganization and cell survival. There are two
isoforms of Abl: Abl1 and Abl2. Abl1 is the form of interest for
this application. It is sometimes called c-Abl or Abl in the
literature, and we refer to it as Abl.
[0004] An off-target activity of a ROCK2 inhibitor on Abl kinase
may be of benefit in treating ophthalmological diseases of retinal
ganglion cells. ROCK2 and Abl are key kinases that regulate
homeostatic balance of the cytoskeleton, and their perturbation and
kinase hyper-activation causes neuronal dysfunction and cell death.
The neuronal cytoskeleton of projection neurons such as retinal
ganglion cells is particularly susceptible to disturbances in
cytoskeletal regulation because of the long axonal process and
requirement for axonal transport. If a retinal ganglion cells was
the size of a Volkswagen Beetle, its axon would be 2 miles
long.
Ophthalmology
[0005] Glaucoma is a disease that affects retinal ganglion cells
(RGCs), and changes at the optic nerve head where the RGC axons
exit the retina are one of the first visual hallmarks of disease
(Quigley. 2016 Annu Rev Vis Sci. 2: p. 235-254). It has been
estimated that glaucoma will affect more than 80 million
individuals worldwide by 2020, with 6-8 million individuals
becoming bilaterally blind. Glaucoma is the second leading cause of
irreversible blindness, one of the most prevalent neurodegenerative
diseases. Glaucoma starts with a loss of peripheral vision and
painlessly progresses slowly, eventually leading to vision loss,
then blindness. Visual loss results from loss of RGCs, and that
reduced aqueous humor drainage through the trabecular meshwork (TM)
and Schlemm's canal is the root cause of ocular hypertension in
glaucoma. In the initial stages, activities involving glare and
dark adaption are affected which impacts driving and mobility;
motor accidents and falls are early consequences of glaucoma. The
total annual economic impact of visual disorders to the healthcare
system for Americans aged 40 years and older is estimated at $35
billion.
[0006] Many forms of glaucoma are associated with elevated
intraocular pressure (IOP) and standard treatment is to reduce IOP
with drugs. Because progression of glaucoma is slow and painless,
noncompliance for daily use of IOP-reducing medications is high.
Side-effects make non-compliance even more likely because there is
no immediate impact when eye drops are not applied. Even with daily
treatments, some patients show continuous progression of glaucoma
despite reaching their lowest achievable IOP (Chang et al. 2012
Ophthal. 119(5): p. 978-986). Failure to keep IOP reduced results
in irreversible damage, and patients do not lose vision until there
is permanent neuronal loss. Lowering intraocular pressure slows the
progression of disease, but lowering IOP does not address the
underlying mechanism of RGC death and optic nerve degeneration.
Therefore, glaucoma is controlled, but never cured by daily use of
available eye drops that reduce IOP.
[0007] There are six classes of topical ocular hypotensive drugs
used to lower IOP. Prostaglandin analogs are the biologically
active metabolites of arachidonic acid and its analogs that are
commonly used to reduce IOP. They may reduce IOP by 27%-33%,
typically require once daily dosing, and are generally associated
with good compliance. Rho kinase (ROCK) inhibitors have potential
to slow blockage of the TM by reducing fibrosis, thereby slowing
RGC death. However, non-specific ROCK inhibitors in clinical
development cause significant hyperemia, a side effect that leads
to non-compliance, although long-term use would be needed to
effectively slow disease progression. Non-specific ROCK inhibitors
have been shown to be neuroprotective, but only by intravitreal
injection (Kitaoka et al. 2014 Brain Res. 1018(1): p. 111-118),
which is not a feasible delivery for repetitive treatment in
humans. Thus, drugs that reduce IOP and slow disease progression
are urgently needed to prevent blindness in glaucoma.
[0008] In the eye, the TM is a mechanosensitive structure that
regulates aqueous humor outflow. Aqueous humor is produced by the
ciliary body epithelium lining, and it drains out of the eye
through the TM into Schlemm's canal and into the episcleral venous
system. Glaucoma is believed to be associated with changes in the
TM that increase deposition of extracellular matrix (ECM) adjacent
to Schlemm's canal (Tektas et al. 2009 Exp Eye Res. 88(4): p.
769-775), a process regulated by ROCK (Pattabiraman, P. P. et al.,
2016, Eur. J. of Pharm., 787: P. 32-42). Hyperactivation of ROCK
may increase deposition ECM in human TM cells, slowing drainage
(Pattabiraman et al. 2014 J Cell Physio. 229(7): p. 927-942). Thus,
ROCK inhibitors that suppress fibrogenic activity of TM cells would
loosen the TM to increase aqueous humor outflow and reduce IOP.
[0009] There are two forms of ROCK that may be implicated in
glaucoma. The TM has both ROCK1 and ROCK2 and RGCs have more ROCK2.
ROCK2 is more important for RGC regeneration (U.S. Pat. No.
7,572,913., 2009). Y-27632 and Fasudil, targeting both ROCKs are
the most widely used reference ROCK inhibitors for research. There
have been 7 different ROCK inhibitors tested in human clinical
trials, most with equal affinity to ROCK1 and ROCK2 (Ren et al.
2016 Invest ophthal Vis Sci. 57(14):p. 6197-6209). Lack of
therapeutic window has hampered the development of ROCK inhibitors,
even when used topically to treat eye diseases (Defert et al. 2017
Expert Opin Ther Pat. 27:507-515).
[0010] ROCK inhibitors may reduce IOP by increasing aqueous humor
outflow through the TM, by contrast to available IOP-reducing drugs
that act on the unconventional pathway of uveoscleral drainage
(Whitlock et al. 2009 J Ocul Pharmacol Ther. 25(3): p. 187-194).
Rho/ROCK pathway is often activated in disease, and they also have
potential to be neuroprotective and increase plasticity and
regeneration of RGC injury. Netarsudil (previously AR-33324) is the
only ROCK inhibitor approved in the USA. Ripasudil is approved in
Japan, but not the USA. Both inhibitors cause hyperemia (red eyes)
as a major side effect (Bacharach et al. 2015 Ophthalmology 122(2):
p. 302-307., Tanihara H. et al. 2016 Acta ophthalmol. 94(1): p.
e26-e34), and therefore patient compliance is expected to be
problematic.
[0011] There is a need for ROCK inhibitors causing reduced or no
hyperemia. There is a need for newdisease-modifying treatments for
glaucoma.
[0012] Retinitis pigmentosa (RP) is a degenerative retinal
dystrophy caused by the progressive degeneration of the rod
photoreceptor cells in the retina. This form of retinal dystrophy
manifests initial symptoms independent of age. The progressive rod
degeneration is followed by abnormalities in the adjacent retinal
pigment epithelium (RPE) and the deterioration of cone
photoreceptor cells. As peripheral vision becomes increasingly
compromised, patients experience progressive "tunnel vision" and
eventual blindness. Affected individuals may additionally
experience defective light-dark adaptations, nyctalopia (night
blindness), and the accumulation of bone spicules in the fundus. RP
is relatively rare inherited disorder that results from mutations
in any one of more than 50 genes required for making proteins that
are needed in functioning photoreceptor cells.
[0013] Macular degeneration, also known as age-related macular
degeneration (AMD or ARMD), is an eye disorder affecting over 235
million people world-wide. Macular degeneration results in blurred
or no vision in the center of the visual field, but does not result
in complete blindness. Visual hallucinations may also occur but
these do not represent a mental illness. Macular degeneration is
the result of damage to the macula of the retina. It may be
age-related, but genetic factors and smoking also play a role. The
severity is divided into early, intermediate, and late types, with
the late type being further divided into "dry" and "wet" forms. The
dry form makes up 90% of cases. Supplements in those who already
have the disease may slow progression, but there is no cure or
treatment that returns vision already lost. In the wet form,
anti-VEGF medication injected into the eye or less commonly laser
coagulation or photodynamic therapy may slow worsening. Targeting
VEGF may reduce pathological growth of blood vessels in the retina
that contribute to pathology of disease.
[0014] There is a need for new therapies for retinitis pigmentosa,
macular degeneration, and retinal angiogenesis.
[0015] Diseases affecting the cornea are a major cause of blindness
worldwide, second only to cataract in overall importance. The
epidemiology of corneal blindness is complicated and encompasses a
wide variety of infectious and inflammatory eye diseases that cause
corneal opacity and scarring, which ultimately leads to functional
blindness. There have been a number of studies that indicate
potential usefulness of ROCK inhibitors for treatment of corneal
diseases that include Fuchs' corneal dystrophy, corneal scarring,
and prevention of scaring complication in glaucoma surgery.
[0016] Fuchs' corneal dystrophy is a progressive, hereditary
disease of the cornea which is late onset and slowly progressing.
Patients often present in the fifth to sixth decade of life with
blurry morning vision that increases in duration as the disease
progresses. Symptoms at presentation include painless decrease in
visual acuity, photophobia, glare and halos around lights. It is a
condition of the posterior cornea and characteristic features
include the formation of focal excrescences of Descemet membrane
termed `guttae`, and loss of endothelial cell density. As disease
advances, corneal edema results in the development of painful
subepithelial and epithelial bullae, and may progress to loss of
corneal sensation, visual acuity and, ultimately, the development
of corneal opacification and pannus formation. The ROCK inhibitor
Y27432 has been used to treat patients with Fuchs membrane
dystrophy. Upon treatment corneal clarity improved and vision
improved for the 24 months the patient was followed (Norika et al
2013. Cornea 32:1167-1170). ROCK inhibitors inhibit
keratocyte-to-myofibroblast transition, and topical application
after a superficial lamellar keratectomy elicits an altered wound
healing response, with evidence of an embryonic-type deposition of
collagen fibrils thus avoiding scar tissue formation in preference
to an ordered regeneration of the wounded tissue (Yamamoto 2012.
Mol Vis. 18:1727-1739).
[0017] In the surgical treatment for glaucoma, the most common
complication of glaucoma surgery is scar formation induced by
activation of a wound healing response that causes fibrosis at the
surgical site. Rho kinase inhibitors reduce activation of human
conjunctival fibroblasts and that treatment with Rho kinase
inhibitor via eyedrops significantly suppresses scar formation
(Futakuchi et al. 2016. Experimental eye research. 149:107-115).
Similarily, BA-1076 will be of therapeutic use in preventing
excessive scarring after glaucoma filtration surgery.
[0018] There is a need for new therapies for the treatment of
corneal blindness, Fuchs' corneal dystrophy, and corneal scarring,
and for reducing post-operative scarring (e.g., post-glaucoma
surgery corneal scarring).
Gastrointestinal Disorders
[0019] Tight junctions are crucial determinants of barrier function
in polarized intestinal epithelia and are significantly regulated
by activity of the Rho-ROCK pathway (Walsh et al.,
Gastroenterology, 2001; 121(3):566). Many conditions can impact
negatively on barrier function in the intestinal epithelium ranging
from inflammation to radiation exposure. It is also known that
inhibition of the Rho-ROCK pathway can limit the activation of
pro-fibrotic pathways, such as are activated in the setting of
inflammatory bowel disorders, and positively impact on paracellular
permeability through tight junctions (Du et al., Gastroent. Res.
Pract.; 2016; 2016: 7374197). Importantly, evidence has also
suggested that inhibition of the c-Abl signaling pathway may also
show anti-fibrotic effects. Having an inhibitor targeting both ROCK
and c-Abl may provide a novel therapeutic approach in this
setting.
[0020] Ionizing radiation can be emitted from atoms of radioactive
isotopes and can be released accidently (e.g., nuclear accident),
by medical procedure (e.g., radiation treatment of cancer) or by
bombs during war. Radiation is a high-energy particle or
electromagnetic radiation that deposits energy when it interacts
with atoms, resulting in ionization (electron excitation). As a
result, an affected cell may either die or malfunction. The
radiation can damage a cell directly by DNA damage, or indirectly
through the creation of unstable, toxic hyperoxide molecules; which
in turn can damage sensitive molecules and afflict subcellular
structures. Radiation damage primarily affect proliferating cells,
and the cell intestine has a very low threshold to radiation damage
because of fast cell turnover. Bone marrow tissue is also
sensitive. Symptoms of acute radiation poisoning are dependent on
the absorbed dose, with symptoms appearing hours to days. There are
treatments for the hematologic disorders that follow radiation
poisoning (e.g., bone marrow transplants, and treatment with G-CSF
(Neupogen). There are no effective treatments for the
gastrointestinal (GI) disorders in ARS.
[0021] The polarized cells epithelial cells of the GI tract that
form a protective barrier against commensal and pathogenic
microorganisms play an important barrier function, in addition to
their role in regulating absorption of nutrients, water, and ion
homeostatic. GI-acute radiation syndrome (ARS) the destruction of
the intestinal epithelial lining causes breakdown of the mucosal
barrier, resulting in diarrhea, dehydration and electrolyte
imbalance. Although all cellular compartments may contribute to and
modulate organ dysfunction, the key event in the pathophysiology of
intestinal radiation toxicity is enterocyte depletion, with
possible vascular damage contributing at higher radiation doses. IN
GI-ARS there is loss of intestinal clonic cells, leading to loss of
epithelia crypts. The severity of mucosal breakdown is dose
dependent, and occurs at radiation levels higher than those that
destroy bone marrow. In the highly polarized epithelial cells of
the GI tract, maintaining the correct balance of active and
inactive ROCK is critical to function of the tissue. Over
activation of Rho cause loss of barrier function because it is a
key regulator of adherens and tight junctions. The ROCK pathway has
been identified as a target of for modulation of intestinal
radiation-induced toxicity (Haydont et al, British Journal of
Radiology, 80 (2007), S32-S40).
SUMMARY
[0022] In general, the invention provides compounds, compositions,
and methods of medical use.
[0023] In one aspect, the invention provides therapeutic
compositions including a therapeutically effective amount of a
compound of formula:
##STR00001##
or a pharmaceutically acceptable salt thereof, where BA-1076 is
stereochemically enriched (e.g., BA-1076 or a pharmaceutically
acceptable salt thereof is present in at least 10% ee, at least 50%
ee, at least 75% ee, at least 80% ee, at least 90% ee, at least 95%
ee, or at least 98% ee). Preferably, BA-1076 or a pharmaceutically
acceptable salt thereof is present in at least 90% ee. More
preferably, BA-1076 or a pharmaceutically acceptable salt thereof
is present in at least 95% ee.
[0024] In another aspect, the invention provides therapeutic
compositions including a therapeutically effective amount of a
compound of formula:
##STR00002##
or a pharmaceutically acceptable salt thereof, where BA-2057 is
stereochemically enriched (e.g., BA-2057 or a pharmaceutically
acceptable salt thereof is present in at least 10% ee, at least 50%
ee, at least 75% ee, at least 80% ee, at least 90% ee, at least 95%
ee, or at least 98% ee). Preferably, BA-2057 or a pharmaceutically
acceptable salt thereof is present in at least 90% ee. More
preferably, BA-2057 or a pharmaceutically acceptable salt thereof
is present in at least 95% ee.
[0025] In some embodiments, the therapeutic composition comprises
BA-1076, or a pharmaceutically acceptable salt thereof, and
BA-2057, or a pharmaceutically acceptable salt thereof. In certain
embodiments, the therapeutic composition is formulated for ocular
topical administration, intravitreal administration, intraocular
administration, retinal administration, oral administration, or
intravenous administration. In further embodiments, the therapeutic
composition is in a dosage form of eye drops. In yet further
embodiments, the therapeutic composition includes the compound at a
concentration of 0.001% to 5% (w/v). In still further embodiments,
the therapeutic composition is formulated for oral administration.
In other embodiments, the therapeutic composition comprises the
compound at a dose of 0.01 mg/kg to 10 mg/kg. In yet other
embodiments, the therapeutic composition is formulated for
intravenous administration. In still other embodiments, the
therapeutic composition comprises the compound at a dose of 0.001
mg/kg to 1 mg/kg. In some embodiments, the therapeutic composition
further includes an IOP-lowering prostaglandin. In particular
embodiments, the IOP-lowering prostaglandin is Travaprost (e.g.,
TRAVATAN.RTM.), Bimatoprost (e.g., LUMIGAN.RTM.), Latanoprost
(e.g., XALATAN.RTM.), or Tafluprost (e.g., ZIOPTAN.RTM.). In
certain embodiments, the prostaglandin analog is Latanoprost (e.g.,
XALATAN.RTM.).
[0026] Inhibitors of ROCK2 (and optionally Abl) described herein
may be useful in treating neurological disorders including
Alzheimer's Disease, Parkinson's Disease, ALS, stroke, and spinal
cord injury and neurotrauma.
[0027] Certain inhibitors of ROCK, alone or in combination with
IOP-lowering prostaglandins, may be useful for treatment of eye
pathologies including glaucoma, retinitis pigmentosa, macular
degeneration, retinal angiogenesis, corneal blindness, Fuchs'
corneal dystrophy, and/or corneal scarring. These ROCK inhibitors
may act by multiple mechanisms to slow disease progression.
[0028] In another aspect, the invention provides a method of
treating Alzheimer's Disease, Parkinson's Disease, ALS, stroke,
spinal cord injury, glaucoma, retinitis pigmentosa, macular
degeneration, retinal angiogenesis, corneal blindness, Fuchs'
corneal dystrophy, or corneal scarring in a subject in need
thereof. Preferably, the method is for treating glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, corneal
blindness, Fuchs' corneal dystrophy, or corneal scarring. In a
related aspect, the invention provides a method of reducing
post-operative corneal scarring (e.g., post-glaucoma surgery
corneal scarring) in a subject in need thereof.
[0029] The methods include, e.g., administering to the subject a
therapeutically effective amount of a therapeutic composition of
the invention (e.g., a therapeutic composition including BA-1076,
or a pharmaceutically acceptable salt thereof, or BA-2057, or a
pharmaceutically acceptable salt thereof). In some embodiments, the
therapeutic composition is administered topically, intravitreally,
intraocularly, retinally to the eye, orally, or intravenously. In
certain embodiments (e.g., in the treatments of glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, corneal
blindness, Fuchs' corneal dystrophy, or corneal scarring), the
therapeutic composition is administered topically to the eye. In
particular embodiments, the therapeutic composition is administered
orally. In further embodiments, the therapeutic composition is
administered intravenously. In yet further embodiments, the method
is for treating glaucoma in the subject. In still further
embodiments, the method is for treating retinitis pigmentosa in the
subject. In other embodiments, the method is for treating macular
degeneration in the subject. In yet other embodiments, the method
is for treating retinal angiogenesis in the subject. In still other
embodiments, the method is for treating corneal blindness. In some
embodiments, the method is for treating Fuchs' corneal dystrophy.
In certain embodiments, the method is for treating corneal
scarring.
[0030] In some embodiments (e.g., in the treatment of glaucoma),
the method further includes administering an IOP-lowering
prostaglandin (e.g., Travaprost (e.g., TRAVATAN.RTM.), Bimatoprost
(e.g., LUMIGAN.RTM., Latanoprost (e.g., XALATAN.RTM.), or
Tafluprost (e.g., ZIOPTAN.RTM.)). In particular embodiments, the
prostaglandin analog is Latanoprost (e.g., XALATAN.RTM.).
[0031] In some embodiments, BA-1076 or a pharmaceutically
acceptable salt thereof is formulated as an oral tablet, e.g., for
daily dosing. In certain embodiments, BA-2057 or a pharmaceutically
acceptable salt thereof is CNS and/or retinal penetrant.
Definitions
[0032] The term "BA-1049," as used herein, refers to a compound of
formula:
##STR00003##
In some embodiments, BA-1049 may be formulated and/or used as a
pharmaceutically acceptable salt. In therapeutic compositions
containing BA-1049, BA-1049 or a pharmaceutically acceptable salt
thereof is stereochemically enriched (e.g., BA-1049 or a
pharmaceutically acceptable salt thereof is present in at least 10%
ee, at least 50% ee, at least 75% ee, at least 80% ee, at least 90%
ee, at least 95% ee, or at least 98% ee). Preferably, BA-1049 or a
pharmaceutically acceptable salt thereof is present in at least 90%
ee. More preferably, BA-1049 or a pharmaceutically acceptable salt
thereof is present in at least 95% ee.
[0033] The term "BA-1076," as used herein, refers to a compound of
formula:
##STR00004##
In some embodiments, BA-1076 may be formulated and/or used as a
pharmaceutically acceptable salt. In some embodiments, BA-1076 may
be formulated and/or used as a pharmaceutically acceptable salt. In
therapeutic compositions containing BA-1076 or a pharmaceutically
acceptable salts thereof, BA-1076 or a pharmaceutically acceptable
salt thereof is stereochemically enriched (e.g., BA-1049 or a
pharmaceutically acceptable salt thereof is present in at least 10%
ee, at least 50% ee, at least 75% ee, at least 80% ee, at least 90%
ee, at least 95% ee, or at least 98% ee). Preferably, BA-1076 or a
pharmaceutically acceptable salt thereof is present in at least 90%
ee. More preferably, BA-1076 or a pharmaceutically acceptable salt
thereof is present in at least 95% ee.
[0034] The term "BA-2017," as used herein, refers to a compound of
formula:
##STR00005##
In some embodiments, BA-2017 may be formulated and/or used as a
pharmaceutically acceptable salt. In some embodiments, BA-2017 may
be formulated and/or used as a pharmaceutically acceptable salt. In
therapeutic compositions containing BA-2017 or a pharmaceutically
acceptable salts thereof, BA-2017 or a pharmaceutically acceptable
salt thereof is stereochemically enriched (e.g., BA-2017 or a
pharmaceutically acceptable salt thereof is present in at least 10%
ee, at least 50% ee, at least 75% ee, at least 80% ee, at least 90%
ee, at least 95% ee, or at least 98% ee). Preferably, BA-2017 or a
pharmaceutically acceptable salt thereof is present in at least 90%
ee. More preferably, BA-2017 or a pharmaceutically acceptable salt
thereof is present in at least 95% ee.
[0035] The term "BA-2057," as used herein, refers to a compound of
formula:
##STR00006##
In some embodiments, BA-2057 may be formulated and/or used as a
pharmaceutically acceptable salt. In some embodiments, BA-2057 may
be formulated and/or used as a pharmaceutically acceptable salt. In
therapeutic compositions containing BA-2057 or a pharmaceutically
acceptable salts thereof, BA-2057 or a pharmaceutically acceptable
salt thereof is stereochemically enriched (e.g., BA-2057 or a
pharmaceutically acceptable salt thereof is present in at least 10%
ee, at least 50% ee, at least 75% ee, at least 80% ee, at least 90%
ee, at least 95% ee, or at least 98% ee). Preferably, BA-2057 or a
pharmaceutically acceptable salt thereof is present in at least 90%
ee. More preferably, BA-2057 or a pharmaceutically acceptable salt
thereof is present in at least 95% ee.
[0036] The term "enantiomeric excess," as used herein, refers to an
art-recognized measure of the proportion of enantiomers in a
composition. Enantiomeric excess is measured in % ee. Percentage
(%) ee can be calculated using the following formula.
( % ) ee = ( C major - C minor ) ( C major + C minor ) 100 % ,
##EQU00001##
where C.sub.major is a molar concentration of the major enantiomer
in a composition, and C.sub.minor is a molar concentration of the
minor enantiomer in the same composition. A composition is
enantiomerically enriched, if (%) ee is greater than 0. A
composition is racemic, if (%) ee is equal to 0.
[0037] The term "IOP-lowering prostaglandin," as used herein,
refers to the biologically active metabolites of arachidonic acid
and their analogs that are commonly used to reduce IOP because of
their effectiveness. IOP-lowering prostaglandins are known in the
art. Non-limiting examples of IOP-lowering prostaglandins include
Travaprost (e.g., TRAVATAN.RTM.), Bimatoprost (e.g., LUMIGAN.RTM.),
Latanoprost (e.g., XALATAN.RTM.), and Tafluprost (e.g.,
ZIOPTAN.RTM.).
[0038] The term "pharmaceutically acceptable salt," as used herein,
represents those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and animals without undue toxicity, irritation, allergic response
and the like and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, pharmaceutically acceptable salts are described in:
Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in
Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H.
Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts can be
prepared in situ during the final isolation and purification of the
compounds described herein or separately by reacting the free base
group with a suitable organic acid. Representative acid addition
salts include acetate, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate,
glucoheptonate, glycerophosphate, hemisulfate, heptonate,
hexanoate, hydrobromide, hydrochloride, hydroiodide,
2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl
sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, stearate, succinate,
sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate,
valerate salts, and the like.
[0039] The term "subject," as used herein, represents a human or
non-human animal (e.g., a mammal) that is suffering from a disease
(e.g., glaucoma, retinitis pigmentosa, macular degeneration,
retinal angiogenesis, Alzheimer's Disease, Parkinson's Disease,
ALS, stroke, or spinal cord injury) or is at risk of a disease
(e.g., glaucoma, retinitis pigmentosa, macular degeneration,
retinal angiogenesis, Alzheimer's Disease, Parkinson's Disease,
ALS, stroke, or spinal cord injury), as determined by a qualified
professional (e.g., a doctor or a nurse practitioner) with or
without known in the art laboratory test(s) of sample(s) from the
patient.
[0040] The terms "treating" or "treat," as used herein, refers to a
therapeutic treatment of a disease (e.g., glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, corneal
blindness, Fuchs' corneal dystrophy, corneal scarring, Alzheimer's
Disease, Parkinson's Disease, ALS, stroke, or spinal cord injury)
in a subject. In some embodiments, a therapeutic treatment may slow
the progression of the disease (e.g., glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, corneal
blindness, Fuchs' corneal dystrophy, corneal scarring, Alzheimer's
Disease, Parkinson's Disease, ALS, stroke, or spinal cord injury),
improve the individual's outcome, and/or eliminate the disease
(e.g., glaucoma, retinitis pigmentosa, macular degeneration,
retinal angiogenesis, corneal blindness, Fuchs' corneal dystrophy,
corneal scarring, Alzheimer's Disease, Parkinson's Disease, ALS,
stroke, or spinal cord injury). In some embodiments, a therapeutic
treatment of a disease (e.g., glaucoma, retinitis pigmentosa,
macular degeneration, retinal angiogenesis, corneal blindness,
Fuchs' corneal dystrophy, corneal scarring, Alzheimer's Disease,
Parkinson's Disease, ALS, stroke, or spinal cord injury) in a
subject may alleviate or ameliorate one or more symptoms or
conditions associated with the disease (e.g., glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, Alzheimer's
Disease, Parkinson's Disease, ALS, stroke, or spinal cord injury),
diminish the extent of the disease (e.g., glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, corneal
blindness, Fuchs' corneal dystrophy, corneal scarring, Alzheimer's
Disease, Parkinson's Disease, ALS, stroke, or spinal cord injury),
stabilize (i.e., not worsening) the state of the disease (e.g.,
glaucoma, retinitis pigmentosa, macular degeneration, retinal
angiogenesis, corneal blindness, Fuchs' corneal dystrophy, corneal
scarring, Alzheimer's Disease, Parkinson's Disease, ALS, stroke, or
spinal cord injury), and/or delay or slow the progress of the
disease (e.g., glaucoma, retinitis pigmentosa, macular
degeneration, retinal angiogenesis, corneal blindness, Fuchs'
corneal dystrophy, corneal scarring, Alzheimer's Disease,
Parkinson's Disease, ALS, stroke, or spinal cord injury), as
compared to the state and/or the condition of the disease (e.g.,
glaucoma, retinitis pigmentosa, macular degeneration, retinal
angiogenesis, corneal blindness, Fuchs' corneal dystrophy, corneal
scarring, Alzheimer's Disease, Parkinson's Disease, ALS, stroke, or
spinal cord injury) in the absence of therapeutic treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The foregoing and other objects of the present disclosure,
the various features thereof, as well as the disclosure itself may
be more fully understood from the following description, when read
together with the accompanying drawings in which:
[0042] FIG. 1 is a graphic representation of the inhibitor profile
of key kinases. Kinases identified in a primary screen with racemic
API were re-tested with 10 .mu.M BA-1076 (closed bars) and BA-1049
(open bars);
[0043] FIGS. 2A, 2B, 2C, and 2D are photographic representations of
hyperemia in the human eye. The photographs show normal (FIG. 2A),
mild (FIG. 2B), medium (FIG. 2C), and severe (FIG. 2D) hyperemia
(from www.aeriepharma.com);
[0044] FIG. 3 is a graph showing intraocular pressure changes,
following the treatment of hypertensive eyes of Cynomolgus monkey
with BA-1076 (racemic) or vehicle. The IOP was measured after a
single application of 1% BA-1076 (racemic, n=9) compared to
untreated lasered eyes (n=7). Reduction in IOP was statistically
significant;
[0045] FIG. 4A is a scheme showing the structure of BA-1076;
[0046] FIG. 4B is a scheme showing the structure of BA-1049;
[0047] FIG. 4C is a scheme showing the structure of BA-2057;
[0048] FIG. 4D is a scheme showing the structure of BA-2017;
[0049] FIG. 5A is an image of an immunoblot showing the dose
response for ROCK inactivation om human trabecular meshwork cells
incubated at predetermined concentrations of BA-1076 or BA-2057.
pMLC is a biomarker of ROCK activation and GAPDH an internal
loading control;
[0050] FIG. 5B is an image of an immunoblot showing the dose
response for ROCK inactivation in human trabecular meshwork cells
incubated at predetermined concentrations of BA-2057 or a
combination of BA-1076 and BA-2057. pMLC is a biomarker of ROCK
activation and GAPDH an internal loading control;
[0051] FIGS. 6A and 6B are images of immunoblots showing the dose
response for ECM deposition in human trabecular meshwork cells
incubated at predetermined concentrations of BA-1076 or BA-2057.
Fibronectin was used is a biomarker of fibrosis and GAPDH an
internal loading control;
[0052] FIG. 7 is a graph showing the neuroprotection of test
compounds one week after optic nerve cut. RGCs were retrogradely
labelled with Fluorogold and counted in retinal whole mounts.
Counts of normal (uninjured) retina are compared with axotomy
alone, PBS injection control, Y-27632 as a comparator ROCK
inhibitor, BA-1076 (racemic) at two different concentrations.
Values shown are means.+-.SEM, n=3-8 animals per group;
[0053] FIGS. 8A and 8B are photomicrographs of adult rat optic
nerve sections showing RGC regeneration after treatment with a ROCK
inhibitor. The rat optic nerves were crushed and treated with 5
.mu.L of 100 .mu.M BA-1049 or vehicle (1.times.PBS) injected in the
vitreous. Axons anterogradely labelled with CTB 2 weeks later
extend past the crush (*). The top photo shows an optic nerve after
injection with vehicle control, the bottom shows an optic nerve
after treatment with BA-1049;
[0054] FIG. 9A is a graph showing the percent reduction in
vascularization of rat eyes treated with BA-1076 (racemic) (left
eye) compared with control eyes treated with PBS (right eye);
[0055] FIG. 9B is a fluorescence micrograph of a BA-1049-treated
eye;
[0056] FIG. 9C is a fluorescence micrograph of a control
PBS-treated eye;
[0057] FIG. 10 is a graph showing the percent vascularization of
rat eyes treated with 0.04 .mu.g, 0.4 .mu.g, or 4.0 .mu.g BA-1076
(racemic);
[0058] FIG. 11 is a graph showing the capability of racemic BA-1076
to slow the progression of retinal degeneration on RD1 mice, as
evidenced by the number of photoreceptors at set distances from the
optic nerve; n=5-7 mice per group. * P.ltoreq.0.05,
**P.ltoreq.0.005
[0059] FIG. 12 is a graph showing the capability of racemic BA-1076
to slow the progression of retinal degeneration on RDS mice, as
evidenced by the number of photoreceptors at set distances from the
optic nerve;
[0060] FIG. 13 is a graph showing the exposure levels of BA-1076
and its metabolite BA-2057 after topical application of a 5%
solution to the eye;
[0061] FIG. 14 is a graphic showing the exposure levels of BA-2017
after topical application of a 3% solution to the eye showing that
the hydroxy metabolite can penetrate ocular tissue after tropical
administration;
[0062] FIG. 15 is a graph showing brain penetrance of BA-1049 and
BA-2017, and high exposure levels in blood vessels;
[0063] FIGS. 16A, 16B, 16C, and 16D are graphs showing BA-1076
IC.sub.50 curves for ROCK1 and ROCK2. The data in FIGS. 16C and 16D
were obtained using higher purity BA-1076 than in FIGS. 16A and
16B;
[0064] FIGS. 16E and 16F are graphs showing BA-2057 IC.sub.50
curves for ROCK1 and ROCK2.
DETAILED DESCRIPTION
[0065] The disclosures of cited herein patents, patent application
publications, and non-patent publications are hereby incorporated
by reference in their entirety in order to more fully describe the
state of the art as known to those skilled in the art as of the
date of the invention described and claimed herein. The instant
disclosure will govern in the instance that there is any
inconsistency between the patents, patent applications, and
publications and this disclosure.
[0066] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs. The
initial definition provided for a group or term herein applies to
that group or term throughout the present specification
individually or as part of another group, unless otherwise
indicated.
[0067] Disclosed are therapeutic compositions including a
therapeutically effective amount of a compound of formula:
##STR00007##
or a pharmaceutically acceptable salt thereof, where BA-1076 or a
pharmaceutically acceptable salt thereof is stereochemically
enriched (e.g., BA-1076 or a pharmaceutically acceptable salt
thereof is present in at least 10% ee, at least 50% ee, at least
75% ee, at least 80% ee, at least 90% ee, at least 95% ee, or at
least 98% ee). Preferably, BA-1076 or a pharmaceutically acceptable
salt thereof is present in at least 90% ee. More preferably,
BA-1076 or a pharmaceutically acceptable salt thereof is present in
at least 95% ee.
[0068] Also disclosed are therapeutic compositions including a
therapeutically effective amount of a compound of formula:
##STR00008##
or a pharmaceutically acceptable salt thereof, where BA-2057 or a
pharmaceutically acceptable salt thereof is stereochemically
enriched (e.g., BA-2057 or a pharmaceutically acceptable salt
thereof is present in at least 10% ee, at least 50% ee, at least
75% ee, at least 80% ee, at least 90% ee, at least 95% ee, or at
least 98% ee). Preferably, BA-2057 or a pharmaceutically acceptable
salt thereof is present in at least 90% ee. More preferably,
BA-2057 or a pharmaceutically acceptable salt thereof is present in
at least 95% ee.
[0069] Surprisingly, BA-1076 was found to inhibit ROCK2 selectively
relative to ROCK1 (see Example 16). Inhibition of ROCK2 is
advantageous in the treatment of eye disorders. Advantageously,
neither BA-1076 nor its metabolite, BA-2057, exhibits off-target
inhibition of GRK1, a rhodopsin kinase involved in phosphorylation
of rhodopsin in mammalian rod cells. In contrast, BA-1049 is
metabolized in vivo to BA-2017, which was found to target GRK1.
Accordingly, unlike BA-1049 or BA-2017, BA-1076 and BA-2057 may be
suitable for the development as a medicament for ophthalmic
applications.
Methods of the Invention
[0070] Without wishing to be bound by theory, ROCK2 is believed to
be active in injured RGCs and in glaucoma (Goldhagen et al. 2012 J
Glau. 21(8): p. 530-538). ROCK may regulate deposition of
extracellular matrix in the TM, and ROCK inhibitors may prevent
ongoing reduction of aqueous outflow by this pathway (Pattabiraman
et al. 2010 Amer J Physiol Cell Physiol. 298(3): p. C749-C763.,
Pattabiraman et al. 2014 J Cell Physiol. 229(7): p. 927-942).
Inhibition of ROCK2 also acts on neurons and simulates plasticity
and regeneration. Inhibition of ROCK may stimulate RGC regeneration
in the optic nerve (Shaw et al. 2016 Exper Eye Res. 158: p. 33-42).
Loss of dendritic connectivity may be one of the earliest event in
glaucoma (EI-Danaf et al. 2015 J Neurosci. 35(6): pm. 2329-2343),
and ROCK inhibitors may stimulate plasticity and connections of
dendrites.
[0071] The invention provides methods of treating a subject in need
thereof, e.g., a subject suffering from glaucoma, retinitis
pigmentosa, macular degeneration, retinal angiogenesis, corneal
blindness, Fuchs' corneal dystrophy, corneal scarring, Alzheimer's
Disease, Parkinson's Disease, ALS, stroke, or spinal cord injury.
The invention also provides a method of reducing post-operative
corneal scarring (e.g., post-glaucoma surgery corneal scarring).
The methods of the invention include administering to the subject
in need thereof a therapeutically effective amount of the
therapeutic composition of the invention (e.g., a therapeutic
composition including BA-1076, or a pharmaceutically acceptable
salt thereof, and/or BA-2057 or a pharmaceutically acceptable salt
thereof).
[0072] The present disclosure also provides a combination therapy
(e.g., a ROCK2 inhibitor (e.g., BA-1076 or BA-2057) in combination
with an IOP-lowering prostaglandin (e.g., latanoprost)) for
treatment of glaucoma, retinitis pigmentosa, macular degeneration,
and retinal angiogenesis and long-term compliance. Existing drugs
successfully control IOP, and ROCK inhibition and treatment with
latanoprost is synergistic in human glaucoma patients (Lewis et al.
2015 Brit J Ophthalmol. 100(3): p. 339-344, Inazaki et al. 2016 J
Glau. 26(2): p. 96-100). A combination therapy is described that
eliminates or reduces the side effect of hyperemia, while improving
the efficacy of outflow through the TM and maintenance of RGC
health while having higher efficacy in reducing IOP. The dose to
impact TM cells and fibrosis may be lower than that required to
lower IOP, and the dose to retain RGC health, as determined by
dendritic arborisation, is less than needed for neuroprotection
and/or regeneration in the optic nerve.
[0073] The combination treatment utilizes a drug that has minimal
side effects to maximize patient compliance and show long-term
benefit in slowing progression of disease.
[0074] Assessment of a compound in the treatment of glaucoma may be
performed in a clinical trial. For example, a glaucoma treatment
clinical trial may include a primary outcome of IOP lowering and
secondary outcome of lack of hyperemia. The same patient population
is followed for a longer period of time post-approval to
investigate reduction of visual loss. The present combination
therapy allows this approach because both dose and off-target
effects contribute to hyperemia.
[0075] Without wishing to be bound by theory, therapeutic
compositions of the invention may slow the progression of glaucoma,
retinitis pigmentosa, macular degeneration, and retinal
angiogenesis because they act on the TM, act on RGCs, and there is
genetic proof of concept that they are a relevant molecular target
in glaucoma (Whitlock et al. 2009 J Ocul Pharmacol Ther. 25(3): p.
187-194). Rho kinases are serine/threonine kinases that regulate
actin/myosin networks within cells. ROCK phosphorylated proteins
directly affect the contractility of the TM and its outflow
properties, and they also regulate the synthesis and deposition of
ECM in the TM (Pattabiraman et al. 2016 Eur J Pharmacol. 787: p.
32-42) ROCK inhibitors promote RGC regeneration, protection and
plasticity (Chang et al. 2012 Ophthalmol. 119(5): p. 979-986, Shaw
et al. 2016 Exper Eye Res. 158: p. 33-42). ROCK inhibition promotes
RGC axon regeneration and protection in vitro and in vivo a finding
now reproduced in many independent labs with Y-27632 and different
inhibitors (Shaw et al. 2016 Exper Eye Res. 158: p. 33-42, Bertrand
et al. 2005 J Neurosci. 25(5): p. 1113-21, Sagawa et al. 2007 Exper
Neurol. 205(1): 9. 230-240) Inhibition of ROCK may maintain
dendritic plasticity in various neurodegenerative diseases, a
process which may be most achievable in the earliest stages of eye
disorders. Inhibition of ROCK is neuroprotective (Shaw et al. 2006
Exper Eye Res. 158: p. 33-42) (FIG. 7). Reversing RGC degeneration
at the earlies stage of the process may be more achievable than
neuroprotection when RGCs are rapidly dying. ROCK inhibitors may
stop VEGF-induced angiogenesis in both macular degeneration and
diabetic retinopathy (van Niew Amerongen et al. 2003 Arterioscler.
Thromb. Vasc. Biol. 23:211-217)
Therapeutic Compositions
[0076] Pharmaceutical formulation is a well-established art, and is
described, e.g., in Gennaro (ed.), Remington. The Science and
Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins
(2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage
Forms and Drug Delivery Systems, 7th ed., Lippincott Williams &
Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.),
Handbook of Pharmaceutical Excipients American Pharmaceutical
Association, 3rd ed. (2000) (ISBN: 091733096X).
[0077] As a therapeutic composition, the ROCK inhibitor compounds
can be mixed with a suitable amount of pharmacologically acceptable
solvent or carrier which are standard in the art for creating
topical eye drops so that to have the appropriate form for
administration to a patient. The term "solvent" relates to diluent,
auxiliary medicinal substance, f or carrier which is mixed with the
ROCK inhibitor(s) for administration to a patient. Similarly, the
term "pharmaceutically-acceptable carrier" includes any and all
solvents, excipients, antibacterial and antifungal agents, and
solutions used in the art to formulate drugs to be applied as eye
drops. The composition can include a pharmaceutically-acceptable
salt (See e.g., Berge et al. (1977) J. Pharm. Sci. 66:1-19).
[0078] Application of the therapeuticcompositions of the present
invention can be both local and/or systemic. For treatment of eye
disease, topical treatment as eye drop is effective. Other
administration methods comprise enteral such as oral, sublingual
and rectal; local such as intraocular, oculo-dermal,
through-dermal, and intradermal; and parenteral. Acceptable
parenteral methods of administration comprise injections, for
example, intravenous, intramuscular, hypodermic injections et
cetera, and non-injection methods. Per ocular or per oral
administration of the compounds and the therapeutic compositions of
the present invention is useful. More specifically, the
administration can be carried out in the form of capsules, tablets,
pills, pillets, granules, syrups, elixirs, solutions,
ophthalmologic solutions, suspensions, emulsions, or
retarded-release substances, or in any other form suitable for
administration to a patient.
[0079] A therapeutically-effective amount of the therapeutic
composition of the invention required for treatment of glaucoma or
related disorder depends on the severity of the disorder or symptom
thereof. The method of administration may be determined at
consultation with a physician such as an ophthalmologist in charge.
In principle, topical solutions range from 0.001% to 5% (w/v)
solution. The eye drops can be applied once daily or up to 3 times
daily. For oral dosing, acceptable doses may be, e.g., from 0.001
mg/kg to 10 mg/kg of a subject's body weight.
[0080] A therapeutically-effective amount of prostaglandin or
prostaglandin analog is the same as the approved range (0.005%
(w/v)) or 10-fold lower (0.0005% (w/v)); ranges for other drugs are
the same as the FDA-approved range or 10-fold lower.
Example 1
Selection of BA-1076 and BA-2057
[0081] ROCK inhibitors are neuroprotective for RGCs in different
models of RGC injury, and they promote axon regeneration in the
optic nerve of adult rats after optic nerve crush (Bertrand et al.
2005 J Neurosci. 25(5): p. 1113-21, Lehmann et al. 1999 J Neurosci.
19(17): p. 7537) One of the earliest changes in glaucoma may be the
loss of RGC connections, both in the retina (EI-Danaf et al. 2015 J
Neurosci. 35(6): p. 2329-2343, Binley et al. 2016 Eur J Neurosci.
44(3): p. 2028-2039) and target areas of the brain (Crish et al.
2010 Proc Natl Acad Sci. 107(11): p. 5196-5201). Neuronal
connectivity is important for RGCs survival and stimulating
regenerative plasticity may be an achievable short-term goal by
acting on RGCs before substantial connection is lost. Once
substantial apoptosis has set in, the process of degeneration may
be more difficult to reverse. The present method restores usable
visual function by regeneration of RGC axons and re-establishing
neural connections in the eye and visual system.
[0082] Molecular modeling and rational drug design were used to
create and screen over 50 ROCK inhibitors. These ROCK inhibitors
were screened for selectivity for ROCK2 and ability to promote
neurite outgrowth. Several lead inhibitors came out of this screen
and further screened for inhibition of key AGC class kinases by a
single dose inhibition assay performed with 10 .mu.M active
pharmaceutical ingredient (API). The lead compound is chiral. The R
and S enantiomers are BA-1049 and BA-1076, respectively. They have
different selectivity for ROCK2 (Table 2) and different off-target
profiles (Table 1; FIG. 1). Neither compound inhibited PKA, thereby
differentiating them from other ROCK inhibitors in development
(FIG. 1).
[0083] A major metabolite of BA-1049, termed BA-2017, is made in
vivo (see US 2017/0313680). BA-2017 was synthesized and determined
to have a cleaner off-target profile than BA-1049 (Table 1).
BA-1076 is also metabolized in vivo to BA-2057. This was
demonstrated by applying BA-1076 topically to rat eyes and
observing the formation of the metabolite in vivo, as detected by
LCMS of collected tissue samples (FIG. 13).
TABLE-US-00001 TABLE 1 DiscoverX Kinome screen. Immobilized kinase
substrates are incubated with DNA-tagged kinase domains tagged plus
API; results are expressed as % of control Tests 10,000 nM API 500
nM performed Kinase or BA- BA- BA- BA- API at the CRO transporter
2017 1049 1076 2057 Netarsudil # Hits <10% 7 11 7 2 11 binding
Kinome ROCK1 >99 >99 >99 >98 93 Screen % ROCK2 >99
>99 >99 >96 93 inhibition PKC .epsilon. 94.7 95 86 <70
93 PKC .DELTA. <85 <70 <70 <70 91 Other GRK1 none (ABL)
none Not known Safety NET none none none none 96 Screen % SERT none
none none none 94 inhibition
[0084] A 468-target kinome screen and a safety screen were carried
out to examine the off-target profiles of different kinase
inhibitors (DiscoverX, Freemont, Calif.). The kinome screen is a
binding assay where API interference of kinase domain/substrate
binding is assessed as percent of control. Less than 10% binding
(or >90% binding inhibition) is most biologically relevant and
considered as a `hit`. BA-2017 only had 7 off-target hits compared
to BA-1049 and Netarsudil, which both had 11 hits (Table 1)
(Studirvant et al. 2016 Bioorg Med Chem Lett. 26(10):2475-2480).
BA-1076 also showed 7 off-target hits, while its active metabolite
BA-2057 showed the cleanest off-target hit profile with only two
hits, which were the target kinases ROCK1 and ROCK2. Published data
for Netarsudil were with 200 times less API. Netarsudil also
inhibits both norepinephrine transporter (NET) and serotonin
transporter (SERT) (Kopczynski et al. 2012 Invest Opthalmol Vis
Sci. 53(14): p. 5080-5080; Studirvant et al. 2016 Bioorg Med Chem
Lett. 26(10):2475-2480). The NET activity likely helps to decrease
IOP but might increase hyperemia, while the SERT activity might
have long-term safety consequences (Costagliola et al. 2004 CNS
drugs 18(8): p. 475-484).
[0085] BA-2017 was found to have the highest affinity for ROCK2
(Table 2). The data in Table 2 were obtained at ATP concentration
below those corresponding to Km.
TABLE-US-00002 TABLE 2 IC50 (.mu.M) determined at 10 .mu.M ATP**
Compound ROCK2 ROCK1 Fold-difference BA-1076 0.73 10 14 BA-1049
0.24 3.9 16 BA-2017 0.05* nd nd BA-2057 nd nd nd *preliminary; nd =
not done**
[0086] A common off-target effect of ROCK inhibitors is inhibition
of PKA because the ATP-binding pocket of ROCK and PKA are highly
conserved (Green et al. 2015 J Med Chem. 58(12): p. 5028-5037).
Y-27632, Fasudil, hydroxyfasudil all bind PKA, and Fasudil binds
ROCK and PKA with same affinity (Jacobs et al. 2006 J Biol Chem.
281(1): p. 260-268). Ripasudil and Netarsudil both inhibit PKA, PKC
and CaMKII (Isobe et al. 2014 Curr Eye Res. 39(8): p. 813-822; Lin
et al. 2018 J Ocul Pharmacol Ther. 34(1-2):40-51). Efficacy and
side effects of ROCK inhibitors are determined by multiple
parameters that include ability to penetrate the cornea,
metabolism, off-target effects, and therefore, in vivo studies are
key to assess safety.
[0087] Oral dosing of 4 cynomolgus monkeys dosed with 18 mg/kg oral
BA-1049 by oral gavage were completed to understand potential
systemic side effects. The only side effects noticed at this dose
was squinting by the monkeys. BA-1049 has some activity toward
G-protein couple protein kinase1, a kinase involved in sensitivity
to light and defects in GRK1 are known to cause Oguchi disease 2
(Orban et al. 2016 G Protein-Couple Receptor Kinases p. 25-43). By
contrast, BA-1076 does not have this off-target effect. Therefore,
this surprising finding shows that BA-1049 is not suitable for
development for treatment of ophthalmological disease, whereas
BA-1076 has an appropriate activity and safety profile.
[0088] Given the selectivity of BA-1076 and BA-2057 for ROCK2, and
the lack of inhibition of kinases important for retinal function,
BA-1076 and BA-2057 were selected as compounds useful for treatment
of ophthalmological disorders.
Example 2
Conjunctival Hyperemia
[0089] A side effect of topical drugs that cause vasodilation is
conjunctival hyperemia. Hyperemia is a serious issue for daily use
and compliance for treatment of glaucoma. Patients object to daily
red eyes, and progression of glaucoma is slow and painless so it is
easy to skip daily dosing. Hyperemia with current ROCK inhibitors
is much higher than with prostaglandins: >50% of treated
patients had hyperemia in the Netarsudil and Ripasudil clinical
studies (Bacharach et al. 2015 Opthalmology 122(2): p. 302-307,
Lewis et al. 2015 Brit J Opthalomo. 100(3): p. 339-344, Tanihara et
al. 2013 Amer J Ophthalmol. 156(4): p. 731-736 e2, Tanihara et al.
2016 Acta Ophthalmol. 94(1): p. e26-e34, Levy et al. 2015 Amer J
Ophthalmol. 159(5): p. 980-958 e1, Tanihara et al. 2013 JAMA
Ophthalmol. 131 (10): p. 1288-1295). BA-1076 did not cause
hyperemia on daily repeat dose study with Dutch Belted rabbits
(Absorption Biosciences) whereas Netarsudil caused 4-8 hours of
mild hyperemia in Dutch belted rabbits (Kopczynski et al. 2012
Invest Opthalmol Vis Sci. 53(14): p. 5080). Hyperemia of Ripasudil
is more frequent and of longer duration than Netarsudil in human
clinical studies (Bacharach et al. 2015 Opthalmology 122(2): p.
302-307, Tanihara et al. 2013 Amer J Ophthalmol. 156(4): p. 731-736
e2)
[0090] To test for hyperemia, the rabbit is a standard species used
in ocular tolerability studies based upon historical data and FDA
requirements. For this study, four (4) Dutch-Belted rabbits
(Oryctolagus cuniculus) were manually restrained to facilitate
topical dosing followed by ocular examinations, and IOP
measurements. Prior to placement on study, each animal underwent an
ophthalmic examination (slit-lamp biomicroscopy, indirect
ophthalmoscopy). Ocular findings were scored according to a
modified McDonald-Shadduck Scoring System. BA-1076 (racemic) or
control article were administered to the animals once daily into
both eyes starting on Day 1. Test and control articles were dosed
in the morning at approximately the same time every day (.about.8
am.+-.2 hours). Animals were observed within their cages once daily
throughout the study period. Animals were observed for changes in
general appearance and behavior. Any abnormal observation was
reported to the Study Director. Ocular findings were scored
according to a modified McDonald-Shadduck Scoring System. The
scoring system 0=normal and numbers of 1-4 score mild to
severe.
[0091] As shown in Table 3, none of the animals treated with
racemic BA-1076 showed any adverse ocular findings.
TABLE-US-00003 TABLE 3 Hyperemia testing in Dutch-Belted rabbits -
Results at Day 3 Vehicle Vehicle BA-1076 BA-1076 Rabbit 1 Rabbit 2
Rabbit 1 Rabbit 2 Eye OD OS OD OD OS OD OS OS Conjunctival 0 0 0 0
0 0 0 0 Discharge Conjunctival 0 0 0 0 0 0 0 0 Congestion
Conjunctival 0 0 0 0 0 0 0 0 Swelling Cornea 0 0 0 0 0 0 0 0
Surface Area of 0 0 0 0 0 0 0 0 Cornea Involvement Pannus 0 0 0 0 0
0 0 0 Pupillary 0 0 0 0 0 0 0 0 Response Aqueous Flare 0 0 0 0 0 0
0 0 Cellular Flare 0 0 0 0 0 0 0 0 Iris Involvement 0 0 0 0 0 0 0
0
[0092] FIG. 2A through FIG. 2D are representative of hyperemia in
the human eye (from www.aeriepharma.com).
[0093] Hyperemia was not correlated with IOP-lowering in a screen
of different ROCK inhibitors (Sturdivant et al. 2016 Bioorg Med
Chem Lett. 26(10): p. 2475-2480), and may be an off-target effect,
or result from different ROCK1/ROCK2 affinity. Vascular endothelial
cells in different tissues vary in ROCK1/ROCK2, and many ROCK
inhibitors target the widely expressed PKA (Green et al. 2015 J Med
Chem. 58(12): p. 5028-5037), a kinase with multiple roles in
cellular homeostasis and response to extracellular signals.
Abnormal activation of the Rho/ROCK pathway, such as occurs in
glaucoma, unbalances the regulation of vascular tone. Blood flow in
the eyes is regulated in large part by vasoactive substances (e.g.,
adenosine and bradykinin) and endothelial-derived nitric
oxide-mediated vasodilation. Thus, multiple mechanisms may
contribute to hyperemia.
[0094] BA-1076 (racemic) was screened for hyperemia in Dutch belted
rabbits for three days of 1% topical dosing. Neither compound
induced any detectable hyperemia. (Table 3). Thus, these ROCK
inhibitors have promise for therapeutic use in treating eye
pathologies.
[0095] Consistent with past trend to report only positive results,
studies with DB rabbits that show IOP lowering by Ripasudil and
Netarsudil did not report effects on hyperemia (Kaneko et al. 2016
Sci rep. 6: Article 19640, Kiel et al. 2014 Invest Ophthalmol Vis
Sci. 55(13): p. 2900-2900). An ARVO poster available on-line shows
that even low doses of Netarsudil (0.04%) caused hyperemia lasting
at least 8 hours on day 1, and it is consistently seen over 10
days, decreases to mild (deLong et al. 2012 Invest. Ophthalmol.
Vis. Sci. 53(14):3867).
Example 3
Combination Therapies
[0096] The present disclosure describes a combination therapy
including a ROCK inhibitor and a prostaglandin or prostaglandin
analog. IOP-lowering prostaglandins are typically biologically
active metabolites of arachidonic acid, or analogs of the
metabolites, that are commonly used to reduce IOP. They can reduce
IOP by 27% to 33% and require only once daily dosing. Such analogs
include Travaprost (e.g., TRAVATAN.RTM.), Bimatoprost (e.g.,
LUMIGAN.RTM.), Latanoprost (e.g., XALATAN.RTM.), or Tafluprost
(e.g., ZIOPTAN.RTM.). This therapy addresses ROCK targets of TM and
RGCs, rather than an inhibitor that will compete with
standard-of-care IOP lowering.
[0097] To determine the efficacy and safety of this combination
therapy, the dose-response of a subject ROCK inhibitor, BA-1076, in
combination with Latanoprost is investigated to achieve exposure of
BA-1076 and its primary metabolite BA-2057 in the TM without
hyperemia, by method described in example 2. Latanoprost is
effective in IOP lowering, and acts synergistically with ROCK
inhibitors (Lewis et al. 2015 Brit J Ophthalmol. 100(3): p.
339-344, Tanihara et al. 2015 JAMA Ophthalmol. 133(7): p. 755-761).
By focusing on biology of TM, retina and therapeutic window, a
combination drug achieves required IOP lowering while providing the
benefits of ROCK inhibition, without the hyperemia.
[0098] A dose of 1% BA-1076 (racemic mixture) was an effective dose
to lower IOP in a monkey model of glaucoma (FIG. 3). It is notable
that there was no significant hyperemia in rabbits or monkeys in
the study of IOP-lowering after topical instillation of BA-1076
(racemic).
Example 4
Activity of Hydroxy Metabolite
[0099] The racemic mixture is composed from the enantiomers BA-1076
and BA-1049 whose structures are shown in FIG. 4A and FIG. 4B,
respectively. These inhibitors have active metabolites, BA-2057
(FIG. 4C) and BA-2017 (FIG. 4D), respectively, which are
enantiomers as well.
[0100] To form the active metabolites BA-2057 and BA-2017 an oxygen
is added to the isoquinoline of the parent compounds BA-1076 or
BA-1049, respectively. The isoquinoline is a structure in common
with Ripasudil. It is likely that aldehyde oxidase (AO) converts
the parent (BA-1076 or BA-1049) to the active metabolite (BA-2057
or BA-2017, respectively). AO is a cytosolic enzyme that has high
expression in brain (Strolin Benedetti et al. 2006 Expert opi drug
metab toxicol. 2(2): p. 895-921), superficial cornea and
choroid-retina (Isobe et al. 2016 J Ocu Pharmacol Ther. 32(7): p.
405-414).
[0101] To examine the potency of BA-1076 an its active metabolite
BA-2057 in disease-relevant cell-based assay, human trabecular
meshwork (TM) cells were cultured and dose-response analysis were
performed. Different concentrations of BA-1076, BA-2057 or a
combination of both were tested.
[0102] To quantify the inhibitory effect of the compounds the TM
cells were serum starved and treated for 1 hour with the indicated
compound concentration and then lysed and extracted for SDS-PAGE
and immunoblotting for phosphorylated myosin light chain kinase 2
(pMLC2), which is a biomarker for cellular ROCK activity.
[0103] These dose-response studies using BA-1076, the active
metabolite BA-2057, or a combination of both on human trabecular
meshwork cells in culture showed potency to reverse ROCK
activation, with the metabolite BA-2057 was more potent than the
parent BA-1076 and (FIG. 5). Combination of both parent and
metabolite further increased potency.
Example 5
Kinome Screening
[0104] Selectively to a broad menu of human kinases of BA-2017,
BA-2057, BA-1049 and BA-1076 was tested using DiscoverX kinome
screen (Table 1). In the kinome screen BA-2017 had fewer off-target
hits (Table 1) than BA-1049. Importantly, BA-1049 showed binding to
G-protein couple protein kinase1 (GRK1) (Table 1) one of 7 GRKs
that phosphorylates rhodopsin and defects in GRK1 function are
known to cause Oguchi disease Type 2. By contrast, BA-1076 does not
have this off-target effect. An interesting hit from the kinome
screen was on Abl (Table 1), an oncogene that also regulates many
cellular activities, including vascular leakage. Although a second
kinome screen did not confirm significant binding of BA-1076 to
Abl, with a potential activity towards Abl, BA-1076 may have
potential to treat retinal diseases with vascular involvement, such
as neovascular glaucoma, diabetic macular edema, and age-related
macular degeneration and be further efficacious in neurological
disorders where both ROCK2 and Abl actively participate in
development of disease including Alzheimer's disease and
Parkinson's disease.
[0105] The data on off-target hits with the R enantiomer (BA-1049)
and the S-enantiomer (BA-1076) that have different biological
activity highlight the surprising finding that BA-1049 is not
suitable for use for treatment of ophthalmological diseases because
it also inactivates a key kinase required for photoreceptor
sensitivity. The finding that BA-1076 inhibits ROCK2 and
potentially Abl indicates the surprising finding that BA-1076 could
be a suitable drug for treatment of neurological diseases where
both kinases are abnormally activated, also because its primary
metabolite BA-2057 showed an even cleaner off-target profile than
BA-2017 (Table 1).
[0106] Effects of BA-1076, BA-2057, Ripasudil (Kowa), and
Netarsudil (Aerie) on the stimulation of hyperemia in Dutch-Belted
(DB) rabbits may be compared. The comparison may be for an acute
hyperemia or chronic hyperemia, e.g., in long term studies.
[0107] A safety screen with BA-1076 and the active metabolite
BA-2057 was completed to check potential agonistic or antagonistic
off-target liability against a broad menu of human targets
important for pharmaceutical safety profiling. These targets
include GPCRs, transporters, ion channels, nuclear receptors,
non-kinase enzymes. Of the 88 targets tested, no significant off
target hits were detected that would confer safety risk.
Example 6
Efficacy on IOP, Aqueous Humor Dynamics
[0108] For further testing BA-1076 and BA-2057, a monkey model is
used because of similar AO metabolism to humans and similar eye
structure.
[0109] A study with a racemic mixture of BA-1076 showed significant
reduction of intraocular pressure in hypertensive Cynomolgus
monkeys 1 hour and 6 hours after a single topical instillation;
longer time points were not examined (FIG. 3). To further
investigate dose and efficacy in Cynomolgus monkey, a single dose
study is carried out with API alone or in combination with
Latanoprost, and compared to latanoprost alone. Clinical studies
show latanoprost acts synergistically with ROCK inhibitors. Aqueous
humor flow and IOP are measured at baseline and 6 hours after
dosing, a time chosen to allow comparison with published aqueous
humor flow. Aqueous humor flow is measured with a scanning
computerized fluorophotometer after applying fluorescein to the
eye. The dosing is in combination with 0.005% Latanoprost, and four
combination doses are tested, with 2 monkeys (4 eyes) in each
group.
Example 7
Efficacy for Reducing Fibrosis in Trabecular Meshwork (TM)
[0110] To investigate potential reduction of TM fibrosis by the
API+Latanoprost, human TM cells are grown to confluency, and the
effect on cell shape is assessed by actin staining. Fibrosis is
characterized by an excessive deposition of extracellular matrix.
To examine the efficacy of BA-1076 and BA-2057 on ECM deposition is
examined by measuring the ECM protein fibronectin in TM cell
lysates by immunoblotting after stimulation with transforming
growth factor beta (TGF.beta.).
[0111] Activation of the canonical TGF.beta. pathway promotes ECM
in cultured TM cells (Inoue-Mochita et al. 2015; PLoS One
10(3):e0120774) and TGF.beta. concentrations are elevated in
glaucomatous eyes in humans (Agarwal et al., 2015; Molecular Vision
21:612-20). TM cells were first serum-starved for 24 hours and then
treated with 2.5 ng/ml TGF.beta. and different concentrations (0,
1, 3, 10, 30, 100 .mu.M, respectively) of either BA-1076 or
BA-2057.
[0112] After 24 hours of treatment cells were lysed and processed
and fibronectin in each sample was revealed by immunoblotting.
Fibronectin levels decreased dose-dependently, with the most
pronounced decrease in the combination of both drugs. Furthermore,
BA-2057 was more potent in decreasing fibronectin deposition than
BA-1076 (FIG. 6).
[0113] In addition, a combination therapy of API (either BA-1076 or
BA-2057) and Latanoprost is tested and compared against latanoprost
alone to determine if there are synergistic effects of the
combination. Latanoprost increases ECM turnover in the TM and
ciliary body through a different pathway than BA-1076/BA-2057
namely by increasing expression of matrix metalloproteinases
(MMPs), which is not affected by treatment with BA-1076 and with
treatment with BA-2057.
[0114] TM cells are treated with 2.5 ng/ml TGF.beta. and with 1
.mu.M of latanoprost alone or a combination 1 latanoprost and API
(either BA-1076 or BA-2057) for 24 hours. After 24 hours of
treatment cells are lysed and processed and fibronectin in each
sample is revealed by immunoblotting. Combination of latanoprost
and either BA-1076, BA-2057 or a combination of both reduces
fibronectin protein levels more strongly than latanoprost alone.
The most pronounced decrease is observed in the combination of
latanoprost, BA-1076 and BA-2057. Furthermore, Latanoprost and
BA-2057 is more potent in decreasing fibronectin deposition than
Latanoprost and BA-1076.
Example 8
RGC Distal Axonopathy and Changes in RGC Cell Soma
[0115] An early hallmark of glaucoma in animal models are defects
in axonal transport (Nickells et al. 2012 Ann Rev of Neurosci. 35:
p. 153-179, Crish et al. 2010 Proc Natl Acad Sci. 107(11): p.
5196-5201). There is a decrease in slow axonal transport after
optic nerve injury (McKeracher et al. 1990 J Neurosci. 10(8): p.
2834-2841) that coincides with a decrease in tubulin mRNA levels in
RGC cell soma (McKerracher et al. 1993 J Neurosci. 13(6): p.
2617-2626). In addition, after optic nerve injury RGCs lose trophic
responsiveness (Pernet et al. 2006 Brain. 129(Pt 4): p. 10147-26)
and hence, even application of BDNF via viral delivery does not
confer long-term RGC survival but only delays RGC cell death (Di
Polo et al. 1998 Proc Natl Acad Sci USA. 95(7): p. 3978-83).
Restoring axonal transport at the earliest phase of glaucoma has
potential to reverse progress of the disease (Crish et al. 2010
Proc Natl Acad Sci. 107(11): p. 5196-5201). Axon constriction at
the optic nerve head is a site of initial axon damage in glaucoma
and is an early event preceding RGC cell death by apoptosis
(Nickells et al. 2012 Ann Rev of Neurosci. 35: p. 153-179).
[0116] Animal models show that RGC death only occurs late in
disease, and that there is a large window between RGC dysfunction
and death (Chang et al. 2012 Ophthalmology. 119(5): p. 979-986).
The different markers of RGC dysfunction are examined in a rat
model of glaucoma where latex microspheres are injected into the
anterior chamber of the eye to block outflow through the TM. In
Sprague Dawley rat RGC loss is 20%-30% over a 4-6 weeks period when
20 .mu.L of beads are injected weekly. This severe model is used to
start to determine the amount of API in the retina 1 hour after
topical installation of the API/latanoprost combination. Rats are
topically dosed with API/Latanoprost for a week after microbead
injection, a time when IOP is elevated. The right eyes serve as
non-glaucomatous controls.
[0117] The beta-3 (BIII) isotype of tubulin is dramatically reduced
in rat after optic nerve injury and increase when RGCs regenerate
in PN grafts (Fournier et al. 1997 J Neurosci. 17(12): p.
4623-4632). BIII tubulin expression is decreased in RGCs in
glaucoma (Soto et al. 2008 J Neurosci. 28(2): p. 548-561). Loss of
BIII tubulin is a biomarker of early RGC degeneration. Ocular
treatment with BA-1076 and BA-2057 prevents the loss of BIII
tubulin immunostaining in glaucoma eyes in comparison to
vehicle-treated eyes. BIII tubulin is observed in radial sections,
and RGCs identified by labeling with Brin3. For quantitative
comparisons, control and treated radial cryostat sections are cut
in the same block and mounted on the same slide, and labeled
together with BIII isotype-specific antibodies. This study reveals
early changes in tubulin expression that correlate with decreased
axonal transport, and demonstrate that ocular dosing with ROCK
inhibitors can reverse this effect.
[0118] ROCK activation is examined in the same microbead model of
glaucoma in rats 4 weeks after daily topical dosing in rats with
left and right eyes injected with beads. API alone or
API+Latanoprost are delivered once daily by 15 .mu.L eye drop
instilled into the left eyes (glaucoma/treated) and right eyes left
untreated (glaucoma/untreated). Retinas and trabecular meshwork are
prepared for Western blots and probed with p-cofilin and phospho
myosin light chain (p-MLC), as biomarkers of ROCK2 activation.
Retinas and TM deriving from untreated glaucoma eyes show high
levels of ROCK activity as demonstrated by high protein levels
phosphorylated cofilin and myosin light chain 2 (MLC2), both
substrates of ROCK. By contrast, glaucoma eyes treated with BA-1076
and BA-2057 do not show elevated p-cofilin nor p-MLC2 levels
demonstrating that ROCK activation is successfully inhibited.
Example 9
Intraretinal Changes
[0119] In glaucoma, loss of RGC terminals may contribute to loss of
retrograde transport and trophic support (Chang et al. 2012
Ophthalmology. 119(5): p. 979-986), and neurotrophic support is
critical for RGC survival (Shen et al. 1999 Neuron. 23: p.
285-295). In the adult rat, retinal ganglion cells (RGCs) die
rapidly when their axons are severed close to the optic disc (FIG.
7), but fewer RGC die when the nerve is cut further away
(Villegas-Perez et al. 1993 J Neurobio. 24: p. 23-36). Thus, RGC
cell death is not simply loss of target innervation, but a change
of trophic responsiveness of RGCs after injury. Intraretinal
connections are needed for RGC survival, and non-neuronal cells,
such as neurotoxic astrocytes and macrophage factors, impact RGC
health and survival.
[0120] To evaluate changes in the RGC dendritic arbor in glaucoma
and the ability of the API to prevent neuronal loss in the retina,
mice are used which carry an enhanced GFP (eGFP) driven by the Hb9
promoter (B6.Cg-Tg(Hlxb9-GFP)1Tmj/J; Jackson Labs), which directs
GFP expression to the on-off direction-sensitive ganglion cell
(ooDSGC) sub-population of retinal ganglion cells (Trenholm et al.
2011 Neuron. 71(4): p. 683-694). These mice allow visualization of
non-overlapping RGCs for unambiguous identification of individual
dendritic arbors. Treated and control glaucoma eyes are examined,
as well as normal eyes 1 month after weekly microbead injection and
daily eye drops. The eyes are fixed by perfusion and prepared for
retinal whole mounts, and the labeled ganglion cells visualized by
fluorescence microscopy. Images are imported into ImageJ and
analyzed for dendritic field area (using the polygon tool to join
points along the perimeter of the field) and subjected to Sholl
analysis using the plugin for Image J to be able to characterize
dendritic branching and complexity of normal, glaucoma/treated and
glaucoma/control RGCs. Treatment with BA-1076 and BA-2057 protect
and/or restore retinal ganglion cell dendritic arbors.
Example 10
Neuroprotection
[0121] Intravitreal injection of 1% racemic BA-1076 after optic
nerve cut is neuroprotective. Topical application of Netarsudil 3
times per day after rat optic nerve crush was neuroprotective, and
these investigators showed reduction in p-cofilin, a biomarker of
ROCK activation not only in retina, but in the optic nerve. These
study methods are repeated to investigate neuroprotection by our
combination therapy (API/Latanoprost) in an optic nerve crush
model. In the first experiment, analogous to dosing of Netarsudil,
test compound is applied topically 3 times daily. Four animals of
each sex are tested per group, and statistics are with pooled
animals for each treatment. Additional experiments powered to
detect sex differences are carried out. During the course of
experiments, animals are monitored daily for clinical signs,
including hyperemia.
[0122] FIG. 8 shows enhanced RGC axon regeneration in an adult rat
after treatment with racemic BA-1076. The optic nerve was crushed
without affecting the ophthalmic artery and racemic BA-1076 was
applied to the eye by intravitreal injection. Control animals were
treated with phosphate-buffered saline (PBS) as vehicle control.
Two weeks later the eye was injected with 0.5% of cholera toxin
beta subunit, an anterograde neuronal tracer that labels neurons
and their processes. Twenty-four hours later the rats were perfused
through the heart with paraformaldehyde and the optic nerve
removed. The optic nerve was mounted in medium, frozen in
isopentane and cryostat sections prepared. The anterograde tracer
was observed by immunofluorescent microscopy, which revealed that
the BA-1076 treated eye showed enhanced regeneration.
Example 11
Effect of ROCK Inhibitors on Vascularization and Angiogenesis
[0123] Inhibitors of ROCK are also known to affect the process of
vascularization. Angiogenesis involves a complex concerted process
which entails disruption of tissue matrix (allowing invasion),
endothelial cell proliferation, migration and tube formation,
orchestrated by local and inflammatory cells and followed by
recruitment of mural cells. Numerous factors participate in
angiogenesis, and some of these play major roles regardless of the
tissue type. Retinal angiogenesis is one type which is one
manifestation of diabetes.
[0124] To determine if BA-1076 (racemic) has an effect on retinal
angiogenesis, an animal (rat) model was used. Rats are born with a
completely avascular retina and the physiological retinal vascular
development takes place in the first couple of weeks of their lives
in a centripetal manner. At P6, 70% of their retina are
physiologically vascularized. Sprague Dawley rat pups were injected
intravitreally at P3 with 5 .mu.l of 4 .mu.g of racemic BA-1076 in
5 rats and the level of vascularization compared between the
racemic BA-1076 treated (left) eye and the PBS injected right eye.
The animals were sacrificed at P5. The eyes were fixed in 4%
paraformaldehyde for 15 minutes at room temperature. Retinas were
dissected and post-fixed in methanol for 10 minutes at -20.degree.
C. The retinas were incubated overnight with TRITC conjugated
lectin griffonia simplicifolia (Sigma-Aldrich) diluted at 1/100 in
1% PBS Triton X-100. After washing, the retinas were mounted,
viewed and photographed with a fluorescence microscope. The total
surface and the surface of the vascularized area were measured
using a computerized image-analysis system (image pro plus).
Statistical analysis was performed using the paired t-test.
[0125] As shown in FIG. 9A and FIG. 9B, eyes treated with racemic
BA-1076 showed statistically significant reduction in
neovascularization.
[0126] In another experiment, the same model was used to examine
the dose-response and doses of 0.04 .mu.g, 0.4 .mu.g, 4 .mu.g, and
10 .mu.g of BA-1076 (racemic) or PBS as vehicle control were tested
in the same animal model. As shown in FIG. 10, all doses of racemic
BA-1076 reduced neovascularization in the treated retina. Thus,
racemic BA-1076 is a useful therapeutic for treating ocular
neovascularization such as that which occurs in diabetes.
Example 12
Retinitis Pigmentosa and Macular Degeneration
[0127] Mice homozygous for the RD1 mutation have an early onset
retinal degeneration due to a mutation of the Pde6b gene encoding
the beta subunit of cGMP-phosphodiesterase in rod photoreceptor.
This mutation leads to toxic accumulation of the second messenger
cGMP in the cell body, which causes photoreceptor cell death by
apoptosis. In humans, a mutation in the same gene has been found to
be responsible for a form of autosomal recessive retinitis
pigmentosa (RP). RP is the most prevalent cause of registered
visual handicap in those of working age in developed countries. In
RD1 mice, degeneration starts around postnatal day 7-day 9 with
complete disappearance of outer nuclear layer after in less than 4
weeks. The inner nuclear layer and the retinal ganglion cells
appear normal but may show slight quantitative reduction. Although
the eyes of the RD1 mice are devoid of normal rods, they retain
some visual capacity but may suffer from night blindness. About 3%
of cones among the visual cells degenerate at a much slower rate
than do rods, so that a few cones are still present at 18 months.
The RD1 mouse is useful as an animal model for retinal
degeneration.
[0128] The ability of BA-1076 (racemic) to slow disease progression
was tested in RD1 mice. For this study, 1 .mu.L of 4 .mu.g BA-1076
(racemic) was injected in the right eye of each mouse at postnatal
day 12 and 1 .mu.L of PBS injected into the left eye. The mice were
euthanized at P15 and the eyes removed. After fixation in Bouin
fixative, the eye specimens from the animals were dehydrated in
graded alcohol series and embedded in paraffin for sectioning.
Retinal sections were cut vertically through the optic disk at 5
.mu.m thickness from nasal to temporal, and then the sections were
stained with Hematoxylin and Eosin for 5 minutes in each stain.
Hematoxylin stains tissue in a deep blue color while Eosin stains
tissue in a deep red color allowing good visualization of the
retinal layers. Retinal sections taken near the optic nerve were
photographed and thickness of ONL or photoreceptor counts were
measured at set distances from the optic nerve. Six to eight
different pictures/animal were measured for each treatment.
[0129] As shown in FIG. 11, BA-1076 (racemic) is neuroprotective
for photoreceptors. Thus, a single intravitreal injection of
racemic BA-1076 increases photoreceptor survival in a severe mouse
model of retinal degeneration.
[0130] The "retinal degeneration" slow (or RDS) mouse is another
useful animal model for human retinal degeneration. RDS is a mouse
strain with a long-studied form of RP that is related to human
mutations within the RDS-peripherin gene accounting for up to 10%
of dominant cases of the disease. This gene normally produces a
complex protein, critical to the function of light transduction by
photoreceptors. The outer segments of the rod and cone
photoreceptor cells of homozygotes fail to develop normally in RDS
mice; eventually these cells degenerate and die. Degeneration in
RDS mice has an early onset and slow progression. Half of the
photoreceptors are lost between day 10 and day 42 and by 3 months
of age, only 2-3 rows of nucleus are left in the ONL. The
photoreceptor cell bodies and synaptic termini are eventually lost
by apoptosis over a period of 12 months.
[0131] The ability of BA-1076 (racemic) to slow disease progression
was tested in RDS mice. For this study, 1 .mu.L of 5 .mu.g BA-1076
(racemic) was weekly injected intravitreally in the right eye of
each mouse and at the same time 1 .mu.L of PBS was injected
intravitreally into the left eye as control. Dosing began when mice
were 1 month old and the eyes were injected weekly for 3 months,
after which the mice were euthanized. The eyes were removed and
fixed in Bouin fixative. The eyes were then dehydrated in graded
alcohol series and embedded in paraffin for sectioning. Retinal
sections were cut vertically through the optic disk at 5 .mu.m
thickness from nasal to temporal, and then the sections were
stained with Hematoxylin and Eosin for 5 minutes in each stain.
Hematoxylin stains tissue a deep blue while Eosin stains a deep red
allowing good visualization of the retinal layers. Retinal sections
taken near the optic nerve were photographed and thickness of ONL
or photoreceptor counts were measured at set distances from the
optic nerve. Six to eight different pictures/animals were measured
for each treatment.
[0132] As shown in FIG. 12, BA-1076 (racemic) is neuroprotective
for photoreceptors in RDS mice. Thus, repeated weekly BA-1076
intravitreal injections increase photoreceptor survival in a mouse
model of slow retinal degeneration.
Example 13
Penetration of Compound in the Retina, Ocular Tissues, the Brain
and Vascular Tissue
[0133] The following experiment demonstrates the penetration and
distribution of BA-1076 and its metabolite BA-2057 in rat ocular
tissues after topical installation in the eye, and after delivery
by oral or IV administration. Adult Sprague Dawley rats (Charles
River Laboratories) were used for this experiment. Rat tissue was
dissected out at 20 min to 48 hours after drug administration, and
the concentration of BA-1076 and BA-2057 in homogenized brain
tissue was measured using LC-MS/MS analysis. One g of tissue was
homogenized in 1 mL of 1.times.PBS. Homogenates were precipitated
by pipetting 200 .mu.L of homogenate into a tube using aseptically
cut off 1000 .mu.L pipette tips to prevent clogging. Samples are
further diluted with 100 .mu.L PBS to aid in precipitation. 900
.mu.L of cold methanol was added to each sample and samples were
vortexed for 5 to 10 seconds. Samples were placed at 4.degree. C.
for 30 to 40 min and then centrifuged at 10,000 g for 15 min at
4.degree. C. The supernatant is collected and stored at -80.degree.
C. until analyzed.
[0134] As shown in FIGS. 13 and 14, BA-1076 and its
hydroxy-metabolite BA-2057 penetrate the tissue of the eye,
including the CNS tissue of the retina. The tissue concentration of
BA-1076 and active metabolite were determined after topical
instillation of 5% solution of BA-1076 in the eye. Twenty minutes
later the various ocular tissues were dissected, as shown in FIG.
13, and the concentration of BA-1076 and BA-2057 measured by LC-MS.
For the optic nerve sample, the concentration is shown per length
of optic nerve (right y-axis). These studies show that the
metabolite is better able to penetrate the optic nerve, and may be
actively transported into the optic nerve by the RGCs in the retina
that are able to metabolize BA-1076 and that project their axons
into the optic nerve. Therefore, drug is available in the retina
and optic nerve for treating these tissues affected in glaucoma and
other retinal diseases.
[0135] In FIG. 14 we examined the ability of metabolite to
penetrate the ocular tissues of the eye after topical instillation
of a 3% solution of metabolite. In comparison with FIG. 13, that
also the metabolite has adequate penetration into ocular and CNS
tissue. Of particular note is the penetration in the retina and
presence in the optic nerve. All compounds were given topically as
eye drops to the eye of adult Sprague Dawley rats. Twenty minutes
later the indicated tissues were removed and the parent compound
and active metabolite were analyzed by LC/MS. These results
demonstrate that BA-1076 and BA-2057 distribute well in ocular
tissues, which suggests these compounds suitable to treat or manage
ocular diseases, disorders, or injuries.
[0136] As shown in FIG. 15, BA-1049, the R enantiomer of BA-1076,
and its active metabolite (BA-2017) were present in the brain after
oral administration, which demonstrates that BA-1049 is able to
cross the blood brain barrier.
[0137] As shown in FIG. 15, BA-1049 and its active metabolite
BA-2017 accumulate at high concentrations in blood vessels after
oral administration. Pathological angiogenesis that occurs in the
retina is a major feature of leading blinding diseases, and
particularly associated with diabetic retinopathy and age-related
macular degeneration.
[0138] As BA-1049 and BA-2017 are enantiomers of BA-1076 and
BA-2057, respectively, these results strongly suggest that BA-1076
and BA-2057 are suitable compounds to treat or manage CNS diseases,
disorders, or injuries as well as vascular diseases or
disorders.
Neurological Diseases
Example 14
In Vitro Effect of Rho Kinase Inhibitors and Abl Kinase Inhibitors
on Dopaminergic Neurons
[0139] The following experiment demonstrates the ability of rho
kinase inhibitors and Abl kinase inhibitors to protect midbrain
dopaminergic neurons in culture from the well-known toxic effects
of the chemical MPP+ (1-methyl-4-phenylpyridinium). MPP+ is a known
dopaminergic neurotoxin. Previous studies have shown that this
compound causes the overactivation of the c-Abl non-receptor
tyrosine kinase which then phosphorylates the Parkin E3 ubiquitin
ligase leading to its inactivation (Ko et al., Proc. Nat. Acad.
Sci. USA; 2010; 107:16691). Additionally, this neurotoxin is also
known to cause the activation of Rho kinase (ROCK) in the brain
when administered to animals (Rodriguez-Perez et al., Neurobiol.
Dis., 2013; 58:209).
[0140] These in vitro experiments are conducted using primary
cultures of embryonic rat midbrain dopaminergic neurons. MPP+ is
known to cause the death of cultured midbrain dopaminergic neurons.
Rho kinase and Abl kinase inhibitors are tested individually at
varying concentrations for their ability to prevent MPP+-induced
cell death.
[0141] Primary cultures of rat midbrain dopaminergic neurons are
established from cells acutely isolated from the ventral midbrain
of embryonic day 14 rats (Charles River; Wilmington, Mass.). The
cells are plated in serum-free conditions (Neurobasal plus B-27
Supplements; Thermo-Fisher; Waltham, Mass.) onto glass coverslips
coated with poly-ornithine and laminin as attachment factors. Cells
are cultured overnight on the coated coverslips in a 37.degree.
C./5% CO2 tissue culture incubator prior to the initiation of the
varying treatments.
[0142] Beginning on day 2 after plating, either vehicle solution or
varying concentrations of BA-1076 or BA-2057 (0.25, 0.5, 1, 5, 10,
20, or 50 .mu.M) are added to the medium of the neuronal cultures.
On day 3 after plating, MPP+(Sigma Chemical; St. Louis, Mo.) is
added to the medium at 20 .mu.M final concentration. The cultures
are allowed to continue for an additional 48 hours. At the end of
the culture period, the cells are washed, and then fixed with 4%
paraformaldehyde solution prior to permeabilization and blocking
with 10% normal goat serum in PBS. The cells are incubated
overnight with antibodies against tyrosine hydroxylase (TH) (rabbit
anti-TH; EMD-Millipore; Billerica, Mass.), a cell-specific marker
of midbrain dopaminergic neurons. The following day, the cultures
are reacted with a fluorescent Cy3-conjugated goat-anti-rabbit
secondary antibody (Jackson ImmunoResearch; West Grove, Pa.). The
coverslips are mounted onto microscope slides along with
VectaShield anti-fade mounting medium with DAPI (Vector Labs) to
visualize the nuclei. Specimens are examined using fluorescence
microscopy and the numbers of TH-positive neurons with non-pyknotic
nuclei are counted in 6 to 8 high power fields per specimen.
[0143] Analysis of the numbers of TH-positive neurons with normal
nuclei demonstrates that treatment with BA-1076 or BA-2057 leads to
a dose-dependent increase in the number of surviving neurons in
culture following exposure of the neurons to MPP+ as compared to
vehicle identifying the ability of BA-1076 and BA-2057 to combat
the toxicity of MPP+.
Example 15
In Vivo Effect of Rho Kinase and Abl Kinase Inhibitors on
Dopaminergic Neurons
[0144] The following experiment demonstrates the ability of certain
rho kinase and Abl kinase inhibitors to protect midbrain
dopaminergic neurons in vivo in the intact mouse from the
well-known toxic effects of the chemical MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). These in vivo
experiments are conducted using adult C57BL/6 mice (a strain
specifically selected because of their sensitivity to MPTP) to
determine the ability of BA-1076 and BA-2057 to protect midbrain
dopaminergic neurons from the cell death typically induced by
administration of MPTP. Previous studies have shown that this
compound causes the overactivation of the c-Abl non-receptor
tyrosine kinase in these neurons, which then phosphorylates the
Parkin E3 ubiquitin ligase leading to its inactivation (Ko et al.,
Proc. Nat. Acad. Sci. USA; 2010; 107:16691). Additionally, this
neurotoxin is also known to cause the activation of Rho kinase
(ROCK) in the brain when administered to animals (Rodriguez-Perez
et al., Neurobiol. Dis., 2013; 58:209).
[0145] Different cohorts of adult C57BL/6 mice (Charles River;
Wilmington, Mass.) (8 to 10 per group) receive intraperitoneal
injections of either vehicle or 30 mg/kg of MPTP (Sigma; St. Louis,
Mo.) dissolved in 0.9% saline for 5 consecutive days. This
sub-acute/chronic treatment regimen typically leads to the death of
40 to 50% of midbrain dopaminergic neurons of the substantia nigra
within 1 to 2 weeks (Meredith and Rademacher; J Parkinsons Dis.;
2011; 1(1):19).
[0146] Beginning one day prior to the first MPTP or vehicle
injection, different cohorts of mice begin treatment with either 10
or 100 mg/kg per day in their drinking water of BA-1076 or BA-2057
or simply water for the extent of the experiment. The cohorts are
carried forward for 14 days. On the final 2 days of the treatment
paradigm, mouse cohorts are subjected to behavioral testing using
the accelerated rotarod on one day or grid-walking test on the
other day. At completion of the final behavioral test, animals are
deeply anesthetized and then perfused transcardially with 4%
paraformaldehyde in phosphate buffer. Brains are removed from the
craniums and post-fixed for 1-2 hours. The brains are then immersed
in 30% sucrose for cryo-protection. Whole coronal sections (20-25
.mu.m thick) through the midbrain region containing the substantia
nigra are obtained for each animal and thaw mounted onto SuperFost
Plus slides (Thermo-Fisher). Individual sections are reacted with
rabbit anti-tyrosine hydroxylase (TH) antibody (EMD-Millipore)
overnight in PBS containing 0.05% Triton X-100 and 10% normal goat
serum. After washing in PBS/Triton-X-100 multiple times, sections
are reacted with HRP-conjugated goat anti-rabbit secondary
antibody. Following another round of washing, the slides are
reacted with Vecta Stain Elite ABC kit for DAB-enhanced peroxidase
detection (Vector Labs; Burlingame, Calif.). Following peroxidase
reaction, the sections are subjected to Nissl staining to label all
cell types as a counterstain. The sections are then coverslipped in
permanent mounting media.
[0147] Sections are examined by visible microscopy and photographic
images captured. The number of TH positive neurons are estimated in
the series of images by unbiased stereological analysis using
Stereo Investigator. Numbers of TH-positive neurons per area of the
substantia nigra are determined and statistical analyses performed
to compare between, control untreated, MPTP/no treatment,
MPTP/inhibitor treatments.
[0148] The analyses demonstrate that MPTP-treated mice receiving
treatment with either BA-1076 or BA-2057 have improved motor
function in behavioral testing as compared to those with no
treatment. Additionally, histological examination of the region of
the substantia nigra indicates that BA-1076 and BA-2057 prevent a
significant percentage of the cell loss due to the neurotoxic
effects of MPTP treatment as a model of Parkinson disease.
Example 16
IC.sub.50 Analysis of BA-1076 versus ROCK1 and ROCK2 to Assess
Selectivity
[0149] The selectivity of BA-1076 for inhibition of ROCK1 and ROCK2
was tested by an enzyme inhibition assay to identify the
concentration at which the compound reduces the enzymatic activity
of the enzymes by 50%, the half-maximal inhibitory concentration or
IC.sub.50. IC.sub.50 determinations for BA-1076 were performed
using a proprietary direct filter-binding radiometric kinase assay
over a 9-point half-log dilution scale at Eurofins Testing (Dundee,
Scotland, UK). ROCK1 and ROCK2 IC.sub.50 determinations were made
using ATP concentrations at the Km ATP of 70 .mu.M and 15 .mu.M for
ROCK1 and ROCK2, respectively. BA-1076 was tested for its
inhibitory effects against ROCK1 and ROCK2 at concentrations
ranging from 100 .mu.M down to 10 nM.
[0150] As shown in FIGS. 16A, 16B, 16C, and 16D, the IC50 plots
against ROCK1 and ROCK2 identify a significant selectivity of
BA-1076 for the ROCK2 isoform, as evidenced by the leftward shift
in the inhibition curve when ROCK2 is compared to ROCK1.
Calculation of the apparent IC50 in this experiment showed that
BA-1076 inhibited ROCK1 with an IC50 of 72,453 nM and inhibited
ROCK2 with an apparent IC50 of 1305 nM.
[0151] BA-1049, BA-2017, and BA-2057 have also been tested in this
assay, and BA-1076 assay was repeated. The data are shown in Table
4.
TABLE-US-00004 TABLE 4 ROCK1 ROCK1/ROCK2 (fold Compound IC.sub.50
(nM) ROCK2 IC.sub.50 (nM) difference) BA-1076 72453 1305 55.5
BA-1076* 19590 322 60.8 BA-1049 26009 599 43.4 BA-2017 3026 183
16.5 BA-2057 23582 491 48.0 *data were obtained for higher purity
BA-1076.
[0152] For the data shown in Table 4, IC50 inhibition curves were
performed at the respective Km for ATP for both ROCK1 and ROCK2.
Performing these assays at the Km of ATP gives a better
representation of the inhibitory activity. Other comparisons where
differing kinases are tested at a single ATP concentration can
potentially misrepresent the comparative efficacy since enzyme
inhibitory activity tested below the Km for ATP of the enzyme,
produces a circumstance where the enzyme is actually not constantly
bound with ATP and the reaction velocity becomes sub-optimal.
Example 17
Ability of BA-1076 and BA-2057 to Reduce Amyloid Beta and
Phosphorylated Tau Levels In Vitro
[0153] Alzheimer's disease is the most common cause of dementia in
the population. Pathologic hallmarks of the disease include the
presence in the brain of beta-amyloid bearing plaques and
neurofibrillary tangles containing hyperphosphorylated
microtubule-associated protein Tau. The 3.times.-Tg AD mouse model
was developed to serve as a rodent model for the development of the
biochemical hallmarks of human Alzheimer's disease. This mouse
strain harbors 3 transgenes encoding mutant Presenilin-1 (PS1),
human Swedish mutant Amyloid Precursor Protein (APP-Swe), and
mutant P301L-Tau and is obtained from Jackson Laboratories (Bar
Harbor, Me.). Primary cultures of cortical neurons from postnatal
day 1 mice are used to assess the ability of BA-1076 and BA-2057 to
limit the production of both beta-amyloid and phosphorylated forms
of Tau. Previous studies have indicated that changes in ROCK2 can
modulate beta-amyloid production in neurons from the 3.times.-Tg AD
mice and that increased ROCK levels are seen in AD brain
(Herskowitz et al.; J. Neurosci.; 2013; 33(49):19086). Similarly,
Cancino et al (Cancino et al.; Neurobiol. Aging; 2011; 32:1249)
identified the role of increased c-Abl kinase activity in
transgenic AD model mice to direct increased phosphorylation of
Tau. Therefore, this experiment will test whether BA-1076 and
BA-2057 would have utility in preventing both the accumulation of
beta-amyloid and hyperphosphorylated Tau in cultured primary
cortical neurons in vitro.
[0154] Whole brain cortices from one day old (P1) 3.times.-Tg AD
mice (Jackson Labs) are dissected and then dissociated using
trypsinization and trituration before being cultured under serum
free conditions (NeuroBasal Medium plus B27 supplements;
Thermo-Fisher) on dishes pre-coated with poly-ornithine and laminin
as growth substrate. The cultures are plated in 24 well tissue
culture plates, with 100 to 200,000 cells per well at plating.
Cultures are maintained for 5 to 7 days, with half-volume medium
changes every other day, before testing of inhibitor treatments.
Cultures are then treated for 24 or 48 hours with 1, 10 or 50 .mu.M
BA-1076 or BA-2057 or vehicle. Protein extracts are prepared in
RIPA buffer and protein concentrations in each extract are
determined using the Pierce BCA assay (Pierce Biotechnology).
Equivalent amounts of protein from each extract are separated on
4-12% polyacrylamide Bis-Tris gels run in MES buffer
(Thermo-Fisher). After transferring to PVDF membranes, the Western
blots are processed to examine the levels of beta-amyloid precursor
protein (.beta.PAPP), the peptide .beta.-amyloid (A.beta.), and
levels of both phosphorylated and total tau protein.
[0155] For these Western blotting experiments, hyperphosphorylated
Tau is detected using antibody AT8-phospho-Ser-202/Thr-205 (Pierce
Biotechnology, Rockford, Ill.); total phosphorylated and
nonphosphorylated tau is detected with Tau5 antibody (Calbiochem,
San Diego, Calif.); total PAPP is detected using monoclonal
antibody 22C11 (EMD-Millipore) and A.beta. peptide levels are
detected using monoclonal antibody 6E10 (BioLegend; San Diego,
Calif.). The form of A.beta. present can be estimated by comparison
to purified A.beta. 1-40, 1-42 peptides (AnaSpec; Fremont, Calif.)
run as standards on companion gels.
[0156] Inhibition of ROCK2 and optionally c-Abl by either BA-1076
or BA-2057 show a dose-dependent reduction in the accumulation of
phosphorylated forms of Tau protein and a reduced production of
A.beta. peptide in cultured cortical neurons from 3.times.-Tg AD
mice.
Example 18
Treatment of Acute Irradiation Syndrome (ARS)
[0157] A mouse model of total body irradiation (TBI) is used to
assess efficacy of BA-1076 and BA-2057 for treating radiation
injury. At day 0, TBI is carried out at a lethal dose of 8.0 Gy
(Harlan mice) or 9.0 Gy (Coats mice)-doses that are expected to
cause death in about 90% of animals within 30 days. Mice receive
oral BA-1076 or IV BA-2057 injections in doses ranging from 0.1
mg/kg to 30 mg/kg at 24 hours, 48 hours, and 72 hours after
irradiation. Mice are monitored for survival up to day 30. During
this period, mice were deprived of all supportive care, including
antibiotics, to increase the stringency of the survival protocol.
Survival is assessed with Kaplan-Meir curves. For GI histopathology
studies, mice are subjected to TBI at 8.0 Gy (Harlan mice) and
administered either placebo, BA-1076 or BA-2057 for three days.
Mice are euthanized at 3 days and GI tissue is paraffin-embedded,
sectioned and immunolabeled with rabbit anti-mouse
leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5),
a GI stem cell marker that is expressed upon GI injury. Exposure to
TBI (8.6 Gy), resulted in substantial jejunal damage 3 days after
irradiation, as evidenced by the widespread expression of LGR5.
Administration of BA-1076 or BA-2057 mitigated radiation-induced
jejunal damage, with no LGR5 expression evident at the optimal,
efficacious dose.
Other Embodiments
[0158] Various modifications and variations of the described
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention that are obvious to those skilled in the art are intended
to be within the scope of the invention.
[0159] Other embodiments are in the claims.
* * * * *
References